@article {pmid34327324,
year = {2021},
author = {Li, S and Zhang, C and Li, J and Yan, L and Wang, N and Xia, L},
title = {Present and future prospects for wheat improvement through genome editing and advanced technologies.},
journal = {Plant communications},
volume = {2},
number = {4},
pages = {100211},
doi = {10.1016/j.xplc.2021.100211},
pmid = {34327324},
issn = {2590-3462},
abstract = {Wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) is one of the most important staple food crops in the world. Despite the fact that wheat production has significantly increased over the past decades, future wheat production will face unprecedented challenges from global climate change, increasing world population, and water shortages in arid and semi-arid lands. Furthermore, excessive applications of diverse fertilizers and pesticides are exacerbating environmental pollution and ecological deterioration. To ensure global food and ecosystem security, it is essential to enhance the resilience of wheat production while minimizing environmental pollution through the use of cutting-edge technologies. However, the hexaploid genome and gene redundancy complicate advances in genetic research and precision gene modifications for wheat improvement, thus impeding the breeding of elite wheat cultivars. In this review, we first introduce state-of-the-art genome-editing technologies in crop plants, especially wheat, for both functional genomics and genetic improvement. We then outline applications of other technologies, such as GWAS, high-throughput genotyping and phenotyping, speed breeding, and synthetic biology, in wheat. Finally, we discuss existing challenges in wheat genome editing and future prospects for precision gene modifications using advanced genome-editing technologies. We conclude that the combination of genome editing and other molecular breeding strategies will greatly facilitate genetic improvement of wheat for sustainable global production.},
}

@article {pmid34326359,
year = {2021},
author = {Rodriguez-Polo, I and Mißbach, S and Petkov, S and Mattern, F and Maierhofer, A and Grządzielewska, I and Tereshchenko, Y and Urrutia-Cabrera, D and Haaf, T and Dressel, R and Bartels, I and Behr, R},
title = {A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {15439},
pmid = {34326359},
issn = {2045-2322},
support = {DZHK-MD16-DPZ//Deutsches Zentrum für Herz-Kreislaufforschung/ ; },
abstract = {Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.},
}

@article {pmid34299081,
year = {2021},
author = {Alghuthaymi, MA and Ahmad, A and Khan, Z and Khan, SH and Ahmed, FK and Faiz, S and Nepovimova, E and Kuča, K and Abd-Elsalam, KA},
title = {Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants.},
journal = {International journal of molecular sciences},
volume = {22},
number = {14},
pages = {},
pmid = {34299081},
issn = {1422-0067},
support = {UHK project VT2019-2021//Department of Chemistry, Faculty of Science, University of Hradec Kralove, 50003 Hradec Kralove, Czech Republic/ ; },
mesh = {*CRISPR-Cas Systems ; Exosomes/*chemistry ; *Gene Editing ; *Genetic Therapy ; Genome, Plant ; Liposomes/*chemistry ; Nanoparticles/*chemistry ; Plants/*genetics ; },
abstract = {Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.},
}

*CRISPR-Cas Systems
Exosomes/*chemistry
*Gene Editing
*Genetic Therapy
Genome, Plant
Liposomes/*chemistry
Nanoparticles/*chemistry
Plants/*genetics

@article {pmid33876950,
year = {2021},
author = {Cereseto, A and Cradick, TJ and Davies, K},
title = {Base Editors Flex Sights on Sickle-Cell Disease.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {166-168},
doi = {10.1089/crispr.2021.29125.kda},
pmid = {33876950},
issn = {2573-1602},
mesh = {*Anemia, Sickle Cell/epidemiology/therapy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}

*Anemia, Sickle Cell/epidemiology/therapy
CRISPR-Cas Systems
*Gene Editing
Humans

@article {pmid33710778,
year = {2021},
author = {Koyanagi, T and Saga, Y and Takahashi, Y and Tamura, K and Yoshiba, T and Takahashi, S and Taneichi, A and Takei, Y and Urabe, M and Mizukami, H and Fujiwara, H},
title = {Knockout of vasohibin-2 reduces tubulin carboxypeptidase activity and increases paclitaxel sensitivity in ovarian cancer.},
journal = {Cancer medicine},
volume = {10},
number = {8},
pages = {2732-2739},
pmid = {33710778},
issn = {2045-7634},
mesh = {Angiogenic Proteins/*genetics ; Antineoplastic Agents, Phytogenic/pharmacology ; CRISPR-Cas Systems ; Carboxypeptidases/genetics/*metabolism ; Cell Culture Techniques/methods ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; Cyclin B1/metabolism ; Female ; Gene Knockdown Techniques ; Humans ; Ovarian Neoplasms/*drug therapy/*genetics/metabolism/pathology ; Paclitaxel/*pharmacology ; Tubulin/metabolism ; Tyrosine/genetics/metabolism ; },
abstract = {Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor, and vasohibin-2 (VASH2), its homolog, exhibits proangiogenic activity. VASH2 is expressed by various cancer cells and accelerates tumor angiogenesis and progression. VASH2 was recently shown to exhibit tubulin carboxypeptidase (TCP) activity related to microtubule functions. Paclitaxel (PTX), an effective chemotherapeutic agent that is widely used to treat ovarian cancer, inhibits microtubule depolymerization and may interact with VASH2. We herein established several VASH2 knockout ovarian cancer cell lines using the CRISPR/Cas9 genome editing system to examine the intracellular tubulin detyrosination status and PTX chemosensitivity. The knockout of VASH2 did not affect the proliferation or sphere-forming activity of ovarian cancer cells in vitro. A Western blot analysis of VASH2 knockout cells revealed the weak expression of detyrosinated tubulin and upregulated expression of cyclin B1. The knockout of VASH2 significantly increased chemosensitivity to PTX, but not to cisplatin in ovarian cancer cell lines. The knockout of VASH2 reduced TCP activity and increased cyclin B1 expression, resulting in increased PTX chemosensitivity in ovarian cancer cells. The inhibition of angiogenesis and regulation of microtubule activity may be achieved in ovarian cancer treatment strategies targeting VASH2.},
}

Angiogenic Proteins/*genetics
Antineoplastic Agents, Phytogenic/pharmacology
CRISPR-Cas Systems
Carboxypeptidases/genetics/*metabolism
Cell Culture Techniques/methods
Cell Line, Tumor
Cell Proliferation/drug effects/genetics
Cyclin B1/metabolism
Female
Gene Knockdown Techniques
Humans
Ovarian Neoplasms/*drug therapy/*genetics/metabolism/pathology
Paclitaxel/*pharmacology
Tubulin/metabolism
Tyrosine/genetics/metabolism

@article {pmid33676891,
year = {2021},
author = {Tian, X and Zhou, B},
title = {Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100509},
pmid = {33676891},
issn = {1083-351X},
mesh = {CRISPR-Cas Systems ; Catalysis ; DNA Nucleotidyltransferases/*metabolism ; Enzyme Activation ; Integrases/metabolism ; Light ; Plants/enzymology ; Promoter Regions, Genetic ; *Recombination, Genetic ; },
abstract = {Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other site-specific recombinase systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation.},
}

CRISPR-Cas Systems
Catalysis
DNA Nucleotidyltransferases/*metabolism
Enzyme Activation
Integrases/metabolism
Light
Plants/enzymology
Promoter Regions, Genetic
*Recombination, Genetic

@article {pmid33058471,
year = {2021},
author = {Xiong, X and Liang, J and Li, Z and Gong, BQ and Li, JF},
title = {Multiplex and optimization of dCas9-TV-mediated gene activation in plants.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {4},
pages = {634-645},
doi = {10.1111/jipb.13023},
pmid = {33058471},
issn = {1744-7909},
support = {//National Transgenic Science and Technology Major Program of China/ ; },
mesh = {Arabidopsis/genetics/metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/genetics ; },
abstract = {Synthetic gene activators consisting of nuclease-dead Cas9 (dCas9) for single-guide RNA (sgRNA)-directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9-TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9-TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA-sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000-fold in rice. Moreover, the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9-TV-refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.},
}

Arabidopsis/genetics/metabolism
CRISPR-Cas Systems/genetics/*physiology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Promoter Regions, Genetic/genetics
RNA, Guide/genetics

@article {pmid32916053,
year = {2020},
author = {Angstenberger, M and de Signori, F and Vecchi, V and Dall'Osto, L and Bassi, R},
title = {Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii.},
journal = {ACS synthetic biology},
volume = {9},
number = {10},
pages = {2840-2850},
pmid = {32916053},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cell Cycle/*genetics ; Cell Nucleus/*genetics ; Chlamydomonas reinhardtii/*genetics ; Chloroplast Proteins/genetics ; DNA End-Joining Repair/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; *Genome, Plant ; *Homologous Recombination ; Membrane Proteins/genetics ; Plant Proteins/genetics ; *Transformation, Genetic ; },
abstract = {In Chlamydomonas reinhardtii, the model organism for eukaryotic green algae and plants, the processes of nuclear transformation and genome editing in particular are still marked by a low level of efficiency, and so intensive work is required in order to create and identify mutants for the investigation of basic physiological processes, as well as the implementation of biotechnological applications. In this work, we show that cell synchronization during the stages of the cell cycle, obtained from long-term cultivation under specific growth conditions, greatly enhances the efficiency of transformation and allows the identification of DNA repair mechanisms that occur preferentially at different stages of the cell cycle. We demonstrate that the transformation of synchronized cells at different times was differentially associated with nonhomologous end joining (NHEJ) and/or homologous recombination (HR), and makes it possible to knock-in specific foreign DNA at the genomic nuclear location desired by exploiting HR. This optimization greatly reduces the overall complexity of the genome editing procedure and creates new opportunities for altering genes and their products.},
}

CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems
Cell Cycle/*genetics
Cell Nucleus/*genetics
Chlamydomonas reinhardtii/*genetics
Chloroplast Proteins/genetics
DNA End-Joining Repair/*genetics
Gene Editing/*methods
Gene Knockout Techniques
*Genome, Plant
*Homologous Recombination
Membrane Proteins/genetics
Plant Proteins/genetics
*Transformation, Genetic

@article {pmid32911927,
year = {2020},
author = {Shen, CC and Lin, MW and Nguyen, BKT and Chang, CW and Shih, JR and Nguyen, MTT and Chang, YH and Hu, YC},
title = {CRISPR-Cas13d for Gene Knockdown and Engineering of CHO Cells.},
journal = {ACS synthetic biology},
volume = {9},
number = {10},
pages = {2808-2818},
doi = {10.1021/acssynbio.0c00338},
pmid = {32911927},
issn = {2161-5063},
mesh = {Animals ; Batch Cell Culture Techniques ; CHO Cells ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cricetulus ; Endonucleases/*genetics ; Fucosyltransferases/genetics ; Gene Expression ; Gene Knockdown Techniques/*methods ; Gene Silencing ; Immunoglobulin G/analysis/biosynthesis ; Metabolic Engineering/*methods ; RNA, Guide/genetics ; Transfection ; },
abstract = {Chinese hamster ovary (CHO) cells are the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism, and glycosylation in CHO cells is desired but challenging. Here, we present a novel approach that exploits CRISPR-Cas13d for gene silencing and CHO cell engineering. CRISPR-Cas13d is a burgeoning system that exploits Cas13d nuclease and guide RNA (gRNA) for RNA cleavage and gene knockdown. We first showed that CRISPR-Cas13d effectively knocked down exogenous genes in CHO cell lines (K1, DG44, and DUXB11) commonly used for recombinant protein production. We next demonstrated that CRISPR-Cas13d robustly suppressed the expression of exogenous genes and various endogenous genes involved in gene amplification, apoptosis, metabolism, and glycosylation (e.g., GS, BAK, BAX, PDK1, and FUT8) in CHO cells with efficiencies ranging from 60% to 80%, simply by transient transfection. By integrating the entire CRISPR-Cas13d system with the Sleeping Beauty system and optimal gRNA design, we further improved the knockdown efficiency and rapidly generated stable cells with ≈80%-90% knockdown. With this approach, we knocked down FUT8 expression for >90% and significantly attenuated the IgG fucosylation. These data altogether implicated the potentials of CRISPR-Cas13d for gene regulation, glycoengineering, and cell engineering of CHO cells.},
}

Animals
Batch Cell Culture Techniques
CHO Cells
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Cricetulus
Endonucleases/*genetics
Fucosyltransferases/genetics
Gene Expression
Gene Knockdown Techniques/*methods
Gene Silencing
Immunoglobulin G/analysis/biosynthesis
Metabolic Engineering/*methods
RNA, Guide/genetics
Transfection

@article {pmid32860515,
year = {2020},
author = {Laudermilk, LT and Tovar, A and Homstad, AK and Thomas, JM and McFadden, KM and Tune, MK and Cowley, DO and Mock, JR and Ideraabdullah, F and Kelada, SNP},
title = {Baseline and innate immune response characterization of a Zfp30 knockout mouse strain.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {31},
number = {7-8},
pages = {205-214},
pmid = {32860515},
issn = {1432-1777},
support = {R01 ES024965/ES/NIEHS NIH HHS/United States ; R01 HL122711/HL/NHLBI NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Biomarkers ; CRISPR-Cas Systems ; Cells, Cultured ; DNA-Binding Proteins/chemistry/*deficiency/genetics/metabolism ; Female ; Gene Editing ; Genetic Association Studies ; Genetic Predisposition to Disease ; Genotype ; Host-Pathogen Interactions/*genetics/*immunology ; Immunity, Innate/*genetics ; Immunomodulation/*genetics ; Male ; Mice ; Mice, Knockout ; Neutrophils/immunology/metabolism ; Phenotype ; Protein Binding ; Protein Interaction Domains and Motifs ; Respiratory Mucosa/immunology/metabolism ; Transcription Factors/chemistry/*deficiency/genetics/metabolism ; },
abstract = {Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.},
}

Alleles
Animals
Biomarkers
CRISPR-Cas Systems
Cells, Cultured
DNA-Binding Proteins/chemistry/*deficiency/genetics/metabolism
Female
Gene Editing
Genetic Association Studies
Genetic Predisposition to Disease
Genotype
Host-Pathogen Interactions/*genetics/*immunology
Immunity, Innate/*genetics
Immunomodulation/*genetics
Male
Mice
Mice, Knockout
Neutrophils/immunology/metabolism
Phenotype
Protein Binding
Protein Interaction Domains and Motifs
Respiratory Mucosa/immunology/metabolism
Transcription Factors/chemistry/*deficiency/genetics/metabolism

@article {pmid32597009,
year = {2020},
author = {Castro Machado, F and Bittencourt-Cunha, P and Malvezzi, AM and Arico, M and Radio, S and Smircich, P and Zoltner, M and Field, MC and Schenkman, S},
title = {EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.},
journal = {Cellular microbiology},
volume = {22},
number = {11},
pages = {e13243},
doi = {10.1111/cmi.13243},
pmid = {32597009},
issn = {1462-5822},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Chagas Disease/*parasitology ; Eukaryotic Initiation Factor-2/genetics/*metabolism ; Gene Expression Regulation ; Humans ; Life Cycle Stages ; Mutation ; Parasitemia ; Phosphorylation ; Protein Biosynthesis ; Proteome/metabolism ; Protozoan Proteins/analysis/biosynthesis/*metabolism ; Trypanosoma cruzi/growth & development/*metabolism/pathogenicity ; Virulence ; },
abstract = {Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.},
}

Animals
CRISPR-Cas Systems
Cell Line
Cell Line, Tumor
Chagas Disease/*parasitology
Eukaryotic Initiation Factor-2/genetics/*metabolism
Gene Expression Regulation
Humans
Life Cycle Stages
Mutation
Parasitemia
Phosphorylation
Protein Biosynthesis
Proteome/metabolism
Protozoan Proteins/analysis/biosynthesis/*metabolism
Trypanosoma cruzi/growth & development/*metabolism/pathogenicity
Virulence

@article {pmid32385892,
year = {2021},
author = {Cai, W and Luo, T and Mao, L and Wang, M},
title = {Spatiotemporal Delivery of CRISPR/Cas9 Genome Editing Machinery Using Stimuli-Responsive Vehicles.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {16},
pages = {8596-8606},
doi = {10.1002/anie.202005644},
pmid = {32385892},
issn = {1521-3773},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Gene Transfer Techniques ; Humans ; },
abstract = {Recent innovations in genome editing have enabled the precise manipulation of the genetic information of mammalians, and benefitted the development of next-generation gene therapy. Despite these advances, several barriers to the clinical translation of genome editing remain, including the intracellular delivery of genome editing machinery, and the risk of off-target editing effect. Here, we review the recent advance of spatiotemporal delivery of CRISPR/Cas9 genome editing machinery, which is composed of programmable Cas9 nuclease and a single-guide RNA (sgRNA) using stimuli-responsive nanoparticles. We discuss the specific chemistries that have been used for controlled Cas9/sgRNA delivery and intracellular release in the presence of endogenous or external signals. These methodologies can leverage biological signals found locally within disease cells, or exogenous signals administrated with spatiotemporal control, through which an improved genome editing could be achieved. We also discuss the future in exploiting these approaches for fundamental biomedical applications and therapeutic genome editing.},
}

Animals
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
*Gene Editing
*Gene Transfer Techniques
Humans

@article {pmid32360447,
year = {2021},
author = {Wang, S and Li, H and Kou, Z and Ren, F and Jin, Y and Yang, L and Dong, X and Yang, M and Zhao, J and Liu, H and Dong, N and Jia, L and Chen, X and Zhou, Y and Qiu, S and Hao, R and Song, H},
title = {Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system.},
journal = {Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases},
volume = {27},
number = {3},
pages = {443-450},
doi = {10.1016/j.cmi.2020.04.018},
pmid = {32360447},
issn = {1469-0691},
mesh = {Antiviral Agents/*pharmacology ; *CRISPR-Cas Systems ; DNA, Viral/*genetics ; Drug Resistance, Viral/*genetics ; Genotype ; Hepatitis B virus/*drug effects/genetics ; Mutation ; Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; },
abstract = {OBJECTIVES: Undetectable or low-level hepatitis B virus (HBV) DNA and drug resistance mutations in patients may increase the risk of HBV transmission or cause active viral replication and other clinical problems. Here, we established a highly sensitive and practical method for HBV and drug resistance detection using a polymerase chain reaction (PCR) -based CRISPR-Cas13a detection system (referred to as PCR-CRISPR) and evaluated its detection capability using clinical samples.

METHODS: Specific CRISPR RNAs (crRNAs) are designed for HBV DNA detection and YMDD (tyrosine-methionine-aspartate-aspartate) variant identification. The HBV DNA was detected in 312 serum samples for HBV diagnosis using quantification PCR (qPCR) and PCR-CRISPR. Additionally, 424 serum samples for YMDD testing were detected by qPCR, direct sequencing, and our assay.

RESULTS: Using PCR-CRISPR, one copy per test of HBV DNA was detected with HBV-1 crRNA in 15 min after PCR amplification. Consistent results with qPCR were observed for 302 samples, while the remaining 10 samples with low-level HBV DNA were detectable by PCR-CRISPR and droplet digital PCR but not by qPCR. PCR-CRISPR diagnosed all 412 drug-resistant samples detected by the YMDD detection qPCR kit and direct sequencing, as well as the other 12 drug-resistant samples with low-level HBV DNA undetectable by qPCR and direct sequencing.

CONCLUSIONS: We developed a novel PCR-CRISPR method for highly sensitive and specific detection of HBV DNA and drug resistance mutations. One copy per test for HBV DNA and YMDD drug resistance mutations could be detected. This method has wide application prospects for the early detection of HBV infection, drug resistance monitoring and treatment guidance.},
}

Antiviral Agents/*pharmacology
*CRISPR-Cas Systems
DNA, Viral/*genetics
Drug Resistance, Viral/*genetics
Genotype
Hepatitis B virus/*drug effects/genetics
Mutation
Polymerase Chain Reaction/*methods
Sensitivity and Specificity

@article {pmid34323910,
year = {2021},
author = {Wang, CS and Chang, CH and Tzeng, TY and Lin, AM and Lo, YL},
title = {Gene-editing by CRISPR-Cas9 in combination with anthracycline therapy via tumor microenvironment-switchable, EGFR-targeted, and nucleus-directed nanoparticles for head and neck cancer suppression.},
journal = {Nanoscale horizons},
volume = {},
number = {},
pages = {},
doi = {10.1039/d1nh00254f},
pmid = {34323910},
issn = {2055-6764},
abstract = {Head and neck cancer (HNC) has a high incidence and a poor prognosis. Epirubicin, a topoisomerase inhibitor, is a potential anthracycline chemotherapeutic for HNC treatment. HuR (ELAVL1), an RNA-binding protein, plays a critical role in promoting tumor survival, invasion, and resistance. HuR knockout via CRISPR/Cas9 (HuR CRISPR) is a possible strategy for the simultaneous modulation of the various pathways of tumor progression. Multifunctional nanoparticles modified with pH-sensitive epidermal growth factor receptor (EGFR)-targeting and nucleus-directed peptides were designed for the efficient delivery of HuR CRISPR and epirubicin to human tongue squamous carcinoma SAS cells and SAS tumor-bearing mice. The pH-sensitive nanoparticles responded to the acidic pH value as a switch to expose the targeting peptides. The cellular uptake and transfection efficiency of these nanoparticles in SAS cells increased via EGFR targeting, ligand-mediated endocytosis, and endosomal escape. These nanoparticles showed low cytotoxicity towards normal oral keratinocyte NOK cells. CRISPR/Cas9 was transported into the nucleus via the nuclear directing peptide and successfully knocked out HuR to suppress proliferation, metastasis, and resistance in SAS cells. The multiple inhibition of EGFR/β-catenin/epithelial-mesenchymal transition pathways was mediated through modulating the EGFR/PI3K/mTOR/AKT axis. The co-treatment of epirubicin and HuR CRISPR in SAS cells further facilitated apoptosis/necroptosis/autophagy and caused cancer cell death. In combination with HuR CRISPR nanoparticles, the efficacy and safety of epirubicin nanoparticles against cancer in SAS tumor-bearing mice improved significantly. Collectively, these nanoparticles showed a tumor pH response, active EGFR targeting, and nuclear localization and thus offered a combinatorial spatiotemporal platform for chemotherapy and the CRISPR/Cas gene-editing system.},
}

@article {pmid34321002,
year = {2021},
author = {Gholizadeh, P and Aghazadeh, M and Ghotaslou, R and Rezaee, MA and Pirzadeh, T and Cui, L and Watanabe, S and Feizi, H and Kadkhoda, H and Kafil, HS},
title = {Role of CRISPR-Cas system on antibiotic resistance patterns of Enterococcus faecalis.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {20},
number = {1},
pages = {49},
pmid = {34321002},
issn = {1476-0711},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are one of the factors which can contribute to limiting the development and evolution of antibiotic resistance in bacteria. There are three genomic loci of CRISPR-Cas in Enterococcus faecalis. In this study, we aimed to assess correlation of the CRISPR-Cas system distribution with the acquisition of antibiotic resistance among E. faecalis isolates. A total of 151 isolates of E. faecalis were collected from urinary tract infections (UTI) and dental-root canal (DRC). All isolates were screened for phenotypic antibiotic resistance. In addition, antibiotic resistance genes and CRISPR loci were screened by using polymerase chain reaction. Genomic background of the isolates was identified by random amplified polymorphic DNA (RAPD)-PCR. The number of multidrug-resistant E. faecalis strains were higher in UTI isolates than in DRC isolates. RAPD-PCR confirmed that genomic background was diverse in UTI and DRC isolates used in this study. CRISPR loci were highly accumulated in gentamycin-, teicoplanin-, erythromycin-, and tetracycline-susceptible strains. In concordance with drug susceptibility, smaller number of CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive strains. These data indicate a negative correlation between CRISPR-cas loci and antibiotic resistance, as well as, carriage of antibiotic resistant genes in both of UTI and DRC isolates.},
}

@article {pmid33970666,
year = {2021},
author = {Akbaba, H and Erel-Akbaba, G and Senturk, S},
title = {Special Focus Issue Part II: Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing.},
journal = {Nanomedicine (London, England)},
volume = {16},
number = {12},
pages = {963-978},
doi = {10.2217/nnm-2020-0412},
pmid = {33970666},
issn = {1748-6963},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Lipids ; *Nanoparticles ; Transfection ; },
abstract = {Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.},
}

CRISPR-Cas Systems/genetics
*Gene Editing
Lipids
*Nanoparticles
Transfection

@article {pmid33664852,
year = {2021},
author = {Chu, CY and Lee, YC and Hsieh, CH and Yeh, CT and Chao, TY and Chen, PH and Lin, IH and Hsieh, TH and Shih, JW and Cheng, CH and Chang, CC and Lin, PS and Huang, YL and Chen, TM and Yen, Y and Ann, DK and Kung, HJ},
title = {Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.},
journal = {Theranostics},
volume = {11},
number = {8},
pages = {3624-3641},
pmid = {33664852},
issn = {1838-7640},
mesh = {Animals ; Arginine/*deficiency ; Argininosuccinate Synthase/deficiency/genetics/metabolism ; Breast Neoplasms/genetics/metabolism/therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chemokine CCL2/genetics/metabolism ; Drug Resistance, Neoplasm/genetics ; Female ; Gene Knockout Techniques ; Humans ; Hydrolases/*pharmacology ; Inflammation/genetics/metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Models, Biological ; Molecular Targeted Therapy ; Polyethylene Glycols/*pharmacology ; Precision Medicine ; Prostatic Neoplasms/genetics/metabolism/therapy ; Signal Transduction ; Triggering Receptor Expressed on Myeloid Cells-1/antagonists & inhibitors/genetics/metabolism ; Up-Regulation ; Xenograft Model Antitumor Assays ; },
abstract = {Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.},
}

Animals
Arginine/*deficiency
Argininosuccinate Synthase/deficiency/genetics/metabolism
Breast Neoplasms/genetics/metabolism/therapy
CRISPR-Cas Systems
Cell Line, Tumor
Chemokine CCL2/genetics/metabolism
Drug Resistance, Neoplasm/genetics
Female
Gene Knockout Techniques
Humans
Hydrolases/*pharmacology
Inflammation/genetics/metabolism
Male
Mice
Mice, Inbred BALB C
Mice, Nude
Models, Biological
Molecular Targeted Therapy
Polyethylene Glycols/*pharmacology
Precision Medicine
Prostatic Neoplasms/genetics/metabolism/therapy
Signal Transduction
Triggering Receptor Expressed on Myeloid Cells-1/antagonists & inhibitors/genetics/metabolism
Up-Regulation
Xenograft Model Antitumor Assays

@article {pmid33420369,
year = {2021},
author = {Jung, H and Lee, KS and Choi, JK},
title = {Comprehensive characterisation of intronic mis-splicing mutations in human cancers.},
journal = {Oncogene},
volume = {40},
number = {7},
pages = {1347-1361},
pmid = {33420369},
issn = {1476-5594},
mesh = {CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Cell Line, Tumor ; Exons/genetics ; Hematologic Neoplasms/*genetics/pathology ; Humans ; Introns/genetics ; Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA Splice Sites/genetics ; RNA Splicing/*genetics ; Telomere-Binding Proteins/*genetics ; },
abstract = {Previous studies studying mis-splicing mutations were based on exome data and thus our current knowledge is largely limited to exons and the canonical splice sites. To comprehensively characterise intronic mis-splicing mutations, we analysed 1134 pan-cancer whole genomes and transcriptomes together with 3022 normal control samples. The ratio-based splicing analysis resulted in 678 somatic intronic mutations, with 46% residing in deep introns. Among the 309 deep intronic single nucleotide variants, 245 altered core splicing codes, with 38% activating cryptic splice sites, 12% activating cryptic polypyrimidine tracts, and 36% and 12% disrupting authentic polypyrimidine tracts and branchpoints, respectively. All the intronic cryptic splice sites were created at pre-existing GT/AG dinucleotides or by GC-to-GT conversion. Notably, 85 deep intronic mutations indicated gain of splicing enhancers or loss of splicing silencers. We found that 64 tumour suppressors were affected by intronic mutations and blood cancers showed higher proportion of deep intronic mutations. In particular, a telomere maintenance gene, POT1, was recurrently mis-spliced by deep intronic mutations in blood cancers. We validated a pseudoexon activation involving a splicing silencer in POT1 by CRISPR/Cas9. Our results shed light on previously unappreciated mechanisms by which noncoding mutations acting on splicing codes in deep introns contribute to tumourigenesis.},
}

CRISPR-Cas Systems/genetics
Carcinogenesis/*genetics
Cell Line, Tumor
Exons/genetics
Hematologic Neoplasms/*genetics/pathology
Humans
Introns/genetics
Mutation/genetics
Polymorphism, Single Nucleotide/genetics
RNA Splice Sites/genetics
RNA Splicing/*genetics
Telomere-Binding Proteins/*genetics

@article {pmid33387638,
year = {2021},
author = {Shirai, Y and Ohde, T and Daimon, T},
title = {Functional conservation and diversification of yellow-y in lepidopteran insects.},
journal = {Insect biochemistry and molecular biology},
volume = {128},
number = {},
pages = {103515},
doi = {10.1016/j.ibmb.2020.103515},
pmid = {33387638},
issn = {1879-0240},
mesh = {Animals ; Biological Evolution ; Bombyx/genetics/metabolism ; CRISPR-Cas Systems ; Genes, Insect ; Insect Proteins/genetics/metabolism ; *Lepidoptera/genetics/metabolism ; Melanins/*metabolism ; Mutagenesis ; Pigmentation/*genetics ; *Spodoptera/genetics/metabolism ; },
abstract = {The diverse colors and patterns found in Lepidoptera are important for success of these species. Similar to the wings of adult butterflies, lepidopteran larvae exhibit diverse color variations to adapt to their habitats. Compared with butterfly wings, however, less attention has been paid to larval body colorations and patterns. In the present study, we focus on the yellow-y gene, which participates in the melanin synthesis pathway. We conducted CRISPR/Cas9-mediated targeted mutagenesis of yellow-y in the tobacco cutworm Spodoptera litura. We analyzed the role of S. litura yellow-y in pigmentation by morphological observation and discovered that yellow-y is necessary for normal black pigmentation in S. litura. We also showed species- and tissue-specific requirements of yellow-y in pigmentation in comparison with those of Bombyx mori yellow-y mutants. Furthermore, we found that almost none of the yellow-y mutant embryos hatched unaided. We provide evidence that S. litura yellow-y has a novel important function in egg hatching, in addition to pigmentation. The present study will enable a greater understanding of the functions and diversification of the yellow-y gene in insects.},
}

Animals
Biological Evolution
Bombyx/genetics/metabolism
CRISPR-Cas Systems
Genes, Insect
Insect Proteins/genetics/metabolism
*Lepidoptera/genetics/metabolism
Melanins/*metabolism
Mutagenesis
Pigmentation/*genetics
*Spodoptera/genetics/metabolism

@article {pmid33323971,
year = {2021},
author = {Periyasamy, M and Singh, AK and Gemma, C and Farzan, R and Allsopp, RC and Shaw, JA and Charmsaz, S and Young, LS and Cunnea, P and Coombes, RC and Győrffy, B and Buluwela, L and Ali, S},
title = {Induction of APOBEC3B expression by chemotherapy drugs is mediated by DNA-PK-directed activation of NF-κB.},
journal = {Oncogene},
volume = {40},
number = {6},
pages = {1077-1090},
pmid = {33323971},
issn = {1476-5594},
support = {12011/CRUK_/Cancer Research UK/United Kingdom ; A18784/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Ataxia Telangiectasia Mutated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Cisplatin/pharmacology ; Cytidine Deaminase/*genetics ; DNA Repair/drug effects ; Etoposide/pharmacology ; Fluorouracil/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Genetic Heterogeneity ; HCT116 Cells ; Humans ; MCF-7 Cells ; Minor Histocompatibility Antigens/*genetics ; Mutation/genetics ; NF-kappa B/genetics ; Neoplasms/*genetics/pathology ; Transcription Factor RelA/*genetics ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.},
}

Ataxia Telangiectasia Mutated Proteins/genetics
CRISPR-Cas Systems/genetics
Cisplatin/pharmacology
Cytidine Deaminase/*genetics
DNA Repair/drug effects
Etoposide/pharmacology
Fluorouracil/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
Genetic Heterogeneity
HCT116 Cells
Humans
MCF-7 Cells
Minor Histocompatibility Antigens/*genetics
Mutation/genetics
NF-kappa B/genetics
Neoplasms/*genetics/pathology
Transcription Factor RelA/*genetics
Tumor Suppressor Protein p53/*genetics

@article {pmid33258137,
year = {2021},
author = {Tian, Z and Wang, JW and Li, J and Han, B},
title = {Designing future crops: challenges and strategies for sustainable agriculture.},
journal = {The Plant journal : for cell and molecular biology},
volume = {105},
number = {5},
pages = {1165-1178},
doi = {10.1111/tpj.15107},
pmid = {33258137},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/*genetics ; Ecosystem ; Gene Editing/*methods ; Genome, Plant/*genetics ; Plant Breeding ; },
abstract = {Crop production is facing unprecedented challenges. Despite the fact that the food supply has significantly increased over the past half-century, ~8.9 and 14.3% people are still suffering from hunger and malnutrition, respectively. Agricultural environments are continuously threatened by a booming world population, a shortage of arable land, and rapid changes in climate. To ensure food and ecosystem security, there is a need to design future crops for sustainable agriculture development by maximizing net production and minimalizing undesirable effects on the environment. The future crops design projects, recently launched by the National Natural Science Foundation of China and Chinese Academy of Sciences (CAS), aim to develop a roadmap for rapid design of customized future crops using cutting-edge technologies in the Breeding 4.0 era. In this perspective, we first introduce the background and missions of these projects. We then outline strategies to design future crops, such as improvement of current well-cultivated crops, de novo domestication of wild species and redomestication of current cultivated crops. We further discuss how these ambitious goals can be achieved by the recent development of new integrative omics tools, advanced genome-editing tools and synthetic biology approaches. Finally, we summarize related opportunities and challenges in these projects.},
}

CRISPR-Cas Systems/genetics
Crops, Agricultural/*genetics
Ecosystem
Gene Editing/*methods
Genome, Plant/*genetics
Plant Breeding

@article {pmid33123984,
year = {2020},
author = {Yang, LQ and Chen, M and Ren, DL and Hu, B},
title = {Dual Oxidase Mutant Retards Mauthner-Cell Axon Regeneration at an Early Stage via Modulating Mitochondrial Dynamics in Zebrafish.},
journal = {Neuroscience bulletin},
volume = {36},
number = {12},
pages = {1500-1512},
pmid = {33123984},
issn = {1995-8218},
mesh = {Animals ; Axons/*physiology ; CRISPR-Cas Systems ; Dual Oxidases/*genetics ; *Mitochondrial Dynamics ; *Nerve Regeneration ; Transcriptome ; *Zebrafish ; },
abstract = {Dual oxidase (duox)-derived reactive oxygen species (ROS) have been correlated with neuronal polarity, cerebellar development, and neuroplasticity. However, there have not been many comprehensive studies of the effect of individual duox isoforms on central-axon regeneration in vivo. Here, we explored this question in zebrafish, an excellent model organism for central-axon regeneration studies. In our research, mutation of the duox gene with CRISPR/Cas9 significantly retarded the single-axon regeneration of the zebrafish Mauthner cell in vivo. Using deep transcriptome sequencing, we found that the expression levels of related functional enzymes in mitochondria were down-regulated in duox mutant fish. In vivo imaging showed that duox mutants had significantly disrupted mitochondrial transport and redox state in single Mauthner-cell axon. Our research data provide insights into how duox is involved in central-axon regeneration by changing mitochondrial transport.},
}

Animals
Axons/*physiology
CRISPR-Cas Systems
Dual Oxidases/*genetics
*Mitochondrial Dynamics
*Nerve Regeneration
Transcriptome
*Zebrafish

@article {pmid32828559,
year = {2021},
author = {Khouzam, JPS and Tivakaran, VS},
title = {CRISPR-Cas9 Applications in Cardiovascular Disease.},
journal = {Current problems in cardiology},
volume = {46},
number = {3},
pages = {100652},
doi = {10.1016/j.cpcardiol.2020.100652},
pmid = {32828559},
issn = {1535-6280},
mesh = {*CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Gene Editing ; Humans ; Mutation ; },
abstract = {The CRISPR-Cas9 system is an economical and accessible gene-editing technology first discovered as a naturally occurring bacterial immune system. Since its fairly recent discovery, CRISPR-Cas9 system's efficiency and simplicity have been successfully used to edit genomes of living organisms in many fields, working in vitro and in vivo in germline and somatic cells to knock-out harmful mutated genes or in some cases working to knock-in a beneficial gene. A current application of the gene-editing system works against specific mutations that cause certain cardiovascular diseases. However, there are current technical limitations as well as ethical dilemmas in introducing gene-editing to humans. Here, we explore highlights on the current state of research of the CRISPR-Cas9 system through the lens of cardiovascular disease and examine potential untouched applications of the system in the field of cardiology.},
}

*CRISPR-Cas Systems
*Cardiovascular Diseases/genetics/therapy
Gene Editing
Humans
Mutation

@article {pmid34314595,
year = {2021},
author = {Boyd, CM and Angermeyer, A and Hays, SG and Barth, ZK and Patel, KM and Seed, KD},
title = {Bacteriophage ICP1: A Persistent Predator of Vibrio cholerae.},
journal = {Annual review of virology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-virology-091919-072020},
pmid = {34314595},
issn = {2327-0578},
abstract = {Bacteriophages or phages-viruses of bacteria-are abundant and considered to be highly diverse. Interestingly, a particular group of lytic Vibrio cholerae-specific phages (vibriophages) of the International Centre for Diarrheal Disease Research, Bangladesh cholera phage 1 (ICP1) lineage show high levels of genome conservation over large spans of time and geography, despite a constant coevolutionary arms race with their host. From a collection of 67 sequenced ICP1 isolates, mostly from clinical samples, we find these phages have mosaic genomes consisting of large, conserved modules disrupted by variable sequences that likely evolve mostly through mobile endonuclease-mediated recombination during coinfection. Several variable regions have been associated with adaptations against antiphage elements in V. cholerae; notably, this includes ICP1's CRISPR-Cas system. The ongoing association of ICP1 and V. cholerae in cholera-endemic regions makes this system a rich source for discovery of novel defense and counterdefense strategies in bacteria-phage conflicts in nature. Expected final online publication date for the Annual Review of Virology, Volume 8 is September 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}

@article {pmid34311897,
year = {2021},
author = {Briggiler Marcó, M and Mercanti, DJ},
title = {Bacteriophages in dairy plants.},
journal = {Advances in food and nutrition research},
volume = {97},
number = {},
pages = {1-54},
doi = {10.1016/bs.afnr.2021.02.015},
pmid = {34311897},
issn = {1043-4526},
abstract = {Bacteriophages represent the main microbiological threat for the manufacture of fermented foods. The dairy industry is the most affected by this problem, as phages are naturally present in raw milk, surfaces, vats, tanks, floors, and distributed by air displacements. Cheese whey may also contain high phage concentrations. Prophages harbored by lysogenic strains could be induced, generating new lytic phages. In this context, where phages cannot be eradicated from dairies, methods of phage monitoring are mandatory. These are mainly based in microbiological features, like classical methods, that are the most used, economic and simple to carry out. Phage DNA detection and quantification by PCR and qPCR, more complex and expensive, are faster, although not able to discern between viable and non-viable virions. Electron microscopy allows direct visualization and characterization of phage morphology, but the apparatus is expensive. Alternative methods based in other phage traits also exist, though less studied and not applicable on a daily basis. Recognition of contamination sources and correct phage monitoring in dairy factories allow a correct application of control measures. These include general measures such as proper factory design, efficient programs of sanitization, good treatment of raw materials, especially milk, and careful handling of by-products. Additionally, the use of starts cultures should be adequate, with application of rotation schemes when possible. Finally, the selection of bacteriophage insensitive mutants (BIM) is essential, and can be achieved simply and empirically, though the study of CRISPR-Cas and other newly discovered mechanisms provide a more rational basis to obtain BIMs with optimized features.},
}

@article {pmid34294769,
year = {2021},
author = {Purusothaman, DK and Shackleford, L and Anderson, MAE and Harvey-Samuel, T and Alphey, L},
title = {CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {14964},
pmid = {34294769},
issn = {2045-2322},
support = {PhD Studentship//The Pirbright Institute/ ; INV-008549//Bill and Melinda Gates Foundation/ ; BBS/E/I/00007033//UK Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BBS/E/I/00007038//UK Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BBS/E/I/00007039//UK Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
abstract = {Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.},
}

@article {pmid34210100,
year = {2021},
author = {Michalski, K and Hertig, C and Mańkowski, DR and Kumlehn, J and Zimny, J and Linkiewicz, AM},
title = {Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8'OH1 loci.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34210100},
issn = {1422-0067},
support = {PB 4-1-03-4-01//POLISH MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT/ ; },
mesh = {CRISPR-Cas Systems ; Cytochrome P-450 Enzyme System/genetics/*metabolism ; Gene Editing/*methods ; Genes, Reporter ; INDEL Mutation ; Mutagenesis, Site-Directed ; Plant Proteins/genetics/*metabolism ; Transfection ; Triticale/genetics/*metabolism ; },
abstract = {Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.},
}

CRISPR-Cas Systems
Cytochrome P-450 Enzyme System/genetics/*metabolism
Gene Editing/*methods
Genes, Reporter
INDEL Mutation
Mutagenesis, Site-Directed
Plant Proteins/genetics/*metabolism
Transfection
Triticale/genetics/*metabolism

@article {pmid33044108,
year = {2020},
author = {Honer, M and Buscemi, K and Barrett, N and Riazati, N and Orlando, G and Nelson, MD},
title = {Orcokinin neuropeptides regulate sleep in Caenorhabditis elegans.},
journal = {Journal of neurogenetics},
volume = {34},
number = {3-4},
pages = {440-452},
pmid = {33044108},
issn = {1563-5260},
support = {P40 OD010440/OD/NIH HHS/United States ; R15 GM122058/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Arthropods/physiology ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/*physiology ; Caenorhabditis elegans Proteins/genetics/*physiology ; Conserved Sequence ; Defecation/physiology ; Gene Editing ; Genes, Helminth ; Hot Temperature ; Loss of Function Mutation ; Motor Activity ; Neurons/metabolism ; Neuropeptides/genetics/*physiology ; Sequence Alignment ; Sleep/genetics/*physiology ; Species Specificity ; Stress, Physiological/physiology ; Transgenes ; Up-Regulation ; },
abstract = {Orcokinin neuropeptides are conserved among ecdysozoans, but their functions are incompletely understood. Here, we report a role for orcokinin neuropeptides in the regulation of sleep in the nematode Caenorhabditis elegans. The C. elegans orcokinin peptides, which are encoded by the nlp-14 and nlp-15 genes, are necessary and sufficient for quiescent behaviors during developmentally timed sleep (DTS) as well as during stress-induced sleep (SIS). The five orcokinin neuropeptides encoded by nlp-14 have distinct but overlapping functions in the regulation of movement and defecation quiescence during SIS. We suggest that orcokinins may regulate behavioral components of sleep-like states in nematodes and other ecdysozoans.},
}

Animals
Animals, Genetically Modified
Arthropods/physiology
CRISPR-Cas Systems
Caenorhabditis elegans/genetics/*physiology
Caenorhabditis elegans Proteins/genetics/*physiology
Conserved Sequence
Defecation/physiology
Gene Editing
Genes, Helminth
Hot Temperature
Loss of Function Mutation
Motor Activity
Neurons/metabolism
Neuropeptides/genetics/*physiology
Sequence Alignment
Sleep/genetics/*physiology
Species Specificity
Stress, Physiological/physiology
Transgenes
Up-Regulation

@article {pmid32772603,
year = {2020},
author = {Liang, JJH and McKinnon, IA and Rankin, CH},
title = {The contribution of C. elegans neurogenetics to understanding neurodegenerative diseases.},
journal = {Journal of neurogenetics},
volume = {34},
number = {3-4},
pages = {527-548},
doi = {10.1080/01677063.2020.1803302},
pmid = {32772603},
issn = {1563-5260},
support = {PJT 165947//CIHR/Canada ; },
mesh = {Animals ; Animals, Genetically Modified ; Apoptosis ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/physiology ; Caenorhabditis elegans Proteins/metabolism ; *Disease Models, Animal ; Genes, Helminth ; Humans ; Mitochondria/metabolism ; Models, Neurological ; Nerve Tissue Proteins/physiology ; Neurodegenerative Diseases/*genetics ; Neurotransmitter Agents/physiology ; Proteostasis ; },
abstract = {Since Caenorhabditis elegans was first introduced as a genetic model organism by Sydney Brenner, researchers studying it have made significant contributions in numerous fields including investigations of the pathophysiology of neurodegenerative diseases. The simple anatomy, optical transparency, and short life-span of this small nematode together with the development and curation of many openly shared resources (including the entire genome, cell lineage and the neural map of the animal) allow researchers using C. elegans to move their research forward rapidly in an immensely collaborative community. These resources have allowed researchers to use C. elegans to study the cellular processes that may underlie human diseases. Indeed, many disease-associated genes have orthologs in C. elegans, allowing the effects of mutations in these genes to be studied in relevant and reproducible neuronal cell-types at single-cell resolution in vivo. Here we review studies that have attempted to establish genetic models of specific human neurodegenerative diseases (ALS, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease) in C. elegans and what they have contributed to understanding the molecular and genetic underpinnings of each disease. With continuous advances in genome engineering, research conducted using this small organism first established by Brenner, Sulston and their contemporaries will continue to contribute to the understanding of human nervous diseases.},
}

Animals
Animals, Genetically Modified
Apoptosis
CRISPR-Cas Systems
Caenorhabditis elegans/*genetics/physiology
Caenorhabditis elegans Proteins/metabolism
*Disease Models, Animal
Genes, Helminth
Humans
Mitochondria/metabolism
Models, Neurological
Nerve Tissue Proteins/physiology
Neurodegenerative Diseases/*genetics
Neurotransmitter Agents/physiology
Proteostasis

@article {pmid32648491,
year = {2020},
author = {Walsh, JD and Boivin, O and Barr, MM},
title = {What about the males? the C. elegans sexually dimorphic nervous system and a CRISPR-based tool to study males in a hermaphroditic species.},
journal = {Journal of neurogenetics},
volume = {34},
number = {3-4},
pages = {323-334},
pmid = {32648491},
issn = {1563-5260},
support = {K12 GM093854/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R01 DK059418/DK/NIDDK NIH HHS/United States ; R01 DK116606/DK/NIDDK NIH HHS/United States ; },
mesh = {Animal Structures/growth & development/innervation/ultrastructure ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*anatomy & histology/genetics/physiology ; Caenorhabditis elegans Proteins/*genetics/physiology ; Cell Cycle Proteins/*genetics ; Cilia/chemistry ; DNA-Binding Proteins/physiology ; Disease Models, Animal ; Extracellular Vesicles/physiology ; Hermaphroditic Organisms/*physiology/ultrastructure ; Humans ; Interneurons/physiology ; Male ; Nervous System/*anatomy & histology/growth & development ; Neuronal Plasticity ; Neurons/classification/physiology/ultrastructure ; Neurotransmitter Agents/physiology ; Nondisjunction, Genetic ; Polycystic Kidney, Autosomal Dominant/genetics ; *Sex Characteristics ; Sexual Behavior, Animal/physiology ; TRPP Cation Channels/genetics ; Transcription Factors/physiology ; },
abstract = {Sexual dimorphism is a device that supports genetic diversity while providing selective pressure against speciation. This phenomenon is at the core of sexually reproducing organisms. Caenorhabditis elegans provides a unique experimental system where males exist in a primarily hermaphroditic species. Early works of John Sulston, Robert Horvitz, and John White provided a complete map of the hermaphrodite nervous system, and recently the male nervous system was added. This addition completely realized the vision of C. elegans pioneer Sydney Brenner: a model organism with an entirely mapped nervous system. With this 'connectome' of information available, great strides have been made toward understanding concepts such as how a sex-shared nervous system (in hermaphrodites and males) can give rise to sex-specific functions, how neural plasticity plays a role in developing a dimorphic nervous system, and how a shared nervous system receives and processes external cues in a sexually-dimorphic manner to generate sex-specific behaviors. In C. elegans, the intricacies of male-mating behavior have been crucial for studying the function and circuitry of the male-specific nervous system and used as a model for studying human autosomal dominant polycystic kidney disease (ADPKD). With the emergence of CRISPR, a seemingly limitless tool for generating genomic mutations with pinpoint precision, the C. elegans model system will continue to be a useful instrument for pioneering research in the fields of behavior, reproductive biology, and neurogenetics.},
}

Animal Structures/growth & development/innervation/ultrastructure
Animals
CRISPR-Cas Systems
Caenorhabditis elegans/*anatomy & histology/genetics/physiology
Caenorhabditis elegans Proteins/*genetics/physiology
Cell Cycle Proteins/*genetics
Cilia/chemistry
DNA-Binding Proteins/physiology
Disease Models, Animal
Extracellular Vesicles/physiology
Hermaphroditic Organisms/*physiology/ultrastructure
Humans
Interneurons/physiology
Male
Nervous System/*anatomy & histology/growth & development
Neuronal Plasticity
Neurons/classification/physiology/ultrastructure
Neurotransmitter Agents/physiology
Nondisjunction, Genetic
Polycystic Kidney, Autosomal Dominant/genetics
*Sex Characteristics
Sexual Behavior, Animal/physiology
TRPP Cation Channels/genetics
Transcription Factors/physiology

@article {pmid34310625,
year = {2021},
author = {Zemmour, A and Dali-Yahia, R and Maatallah, M and Saidi-Ouahrani, N and Rahmani, B and Benhamouche, N and Al-Farsi, HM and Giske, CG},
title = {High-risk clones of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital Establishment of Oran, Algeria (2011-2012).},
journal = {PloS one},
volume = {16},
number = {7},
pages = {e0254805},
doi = {10.1371/journal.pone.0254805},
pmid = {34310625},
issn = {1932-6203},
abstract = {The purpose of the study was to characterize the resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum β-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and to determine their phylogenetic relatedness. The isolates were from Algeria, isolated at the University Hospital Establishment of Oran, between 2011 and 2012. ESBL-KP isolates (n = 193) were screened for several antibiotic resistance genes (ARGs) using qPCR followed by Pulsed-Field Gel Electrophoresis (PFGE). Representative isolates were selected from PFGE clusters and subjected to whole-genome sequencing (WGS). Genomic characterization of the WGS data by studying prophages, CRISPR-Cas systems, Multi-Locus Sequence Typing (MLST), serotype, ARGs, virulence genes, plasmid replicons, and their pMLST. Phylogenetic and comparative genomic were done using core genome MLST and SNP-Based analysis. Generally, the ESBL-KP isolates were polyclonal. The whole genome sequences of nineteen isolates were taken of main PFGE clusters. Sixteen sequence types (ST) were found including high-risk clones ST14, ST23, ST37, and ST147. Serotypes K1 (n = 1), K2 (n = 2), K3 (n = 1), K31 (n = 1), K62 (n = 1), and K151 (n = 1) are associated with hyper-virulence. CRISPR-Cas system was found in 47.4%, typed I-E and I-E*. About ARGs, from 193 ESBL-KP, the majority of strains were multidrug-resistant, the CTX-M-1 enzyme was predominant (99%) and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes was high with aac(6')-lb-cr (72.5%) and qnr's (65.8%). From 19 sequenced isolates we identified ESBL, AmpC, and carbapenemase genes: blaCTX-M-15 (n = 19), blaOXA-48 (n = 1), blaCMY-2 (n = 2), and blaCMY-16 (n = 2), as well as non-ESBL genes: qnrB1 (n = 12), qnrS1 (n = 1) and armA (n = 2). We found IncF, IncN, IncL/M, IncA/C2, and Col replicon types, at least once per isolate. This study is the first to report qnrS in ESBL-KP in Algeria. Our analysis shows the concerning co-existence of virulence and resistance genes and would support that genomic surveillance should be a high priority in the hospital environment.},
}

@article {pmid34310056,
year = {2021},
author = {He, C and Liu, H and Chen, D and Xie, WZ and Wang, M and Li, Y and Gong, X and Yan, W and Chen, LL},
title = {CRISPR-Cereal: A Guide RNA Design Tool Integrating Regulome and Genomic Variation for Wheat, Maize and Rice.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13675},
pmid = {34310056},
issn = {1467-7652},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) genome editing system (CRISPR-Cas) is revolutionizing agriculture. In this system, a guide sequence that matches to a particular genomic DNA is placed in front of a synthetic RNA that consists of a scaffold sequence necessary for Cas-binding to form a guide RNA (gRNA). gRNA/Cas complex binds to the target DNA that contains a protospacer adjacent motif (PAM) via base-paring and generates a double strand break (DSB) by Cas protein. Mutations will be created when the DSB cannot be perfectly repaired. Among kinds of Cas variants, Cas9 and Cas12a (also termed Cpf1) are the two major nucleases with highest edit efficiency. NGG (N=A, T, G or C) for SpCas9 from Streptococcus pyogenes, TTTN for Cpf1 from Acidaminococcus or Lachnospiraceae, is necessary for recruiting the nuclease to produce DSBs.},
}

@article {pmid34305865,
year = {2021},
author = {You, Y and Zhang, P and Wu, G and Tan, Y and Zhao, Y and Cao, S and Song, Y and Yang, R and Du, Z},
title = {Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {700016},
pmid = {34305865},
issn = {1664-302X},
abstract = {The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT-LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 μl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.},
}

@article {pmid34305843,
year = {2021},
author = {Ramachandran, V and Weiland, JJ and Bolton, MD},
title = {CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {679994},
pmid = {34305843},
issn = {1664-302X},
abstract = {Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.},
}

@article {pmid34304411,
year = {2021},
author = {Tan, JJ and Peng, YZ and Huang, GT},
title = {[Research advances on the development and application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein system].},
journal = {Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns},
volume = {37},
number = {7},
pages = {681-687},
doi = {10.3760/cma.j.cn501120-20200329-00201},
pmid = {34304411},
issn = {1009-2587},
support = {81801916, 81960353//National Natural Science Foundation of China for Youth/ ; 20204Y003//Science and Technology Support Project of Guizhou Province of China/ ; },
mesh = {*CRISPR-Associated Proteins ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Nucleic Acids ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein system, as an emerging gene editing system, can be divided into class 1 and class 2 systems according to the number of Cas protein. The CRISPR/Cas9 in class 2 system can cleave target nucleic acid only with the help of Cas9 protein and single-stranded guide RNA, which is currently the most widely used CRISPR/Cas system. In addition to gene editing in the treatment of genetic diseases, a variety of CRISPR/Cas system derived technologies have vast application prospect in the fields of disease-related gene screening, gene expression regulation, and rapid detection, prevention, and control of pathogens. This article summarizes the discovery process of CRISPR/Cas system and applications of several major CRISPR/Cas derived technologies, aiming to provide a reference for researchers in the field of life science.},
}

*CRISPR-Associated Proteins
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
*Nucleic Acids

@article {pmid34304349,
year = {2021},
author = {Okoli, AS and Blix, T and Myhr, AI and Xu, W and Xu, X},
title = {Sustainable use of CRISPR/Cas in fish aquaculture: the biosafety perspective.},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {34304349},
issn = {1573-9368},
abstract = {Aquaculture is becoming the primary source of seafood for human diets, and farmed fish aquaculture is one of its fastest growing sectors. The industry currently faces several challenges including infectious and parasitic diseases, reduced viability, fertility reduction, slow growth, escapee fish and environmental pollution. The commercialization of the growth-enhanced AquAdvantage salmon and the CRISPR/Cas9-developed tilapia (Oreochromis niloticus) proffers genetic engineering and genome editing tools, e.g. CRISPR/Cas, as potential solutions to these challenges. Future traits being developed in different fish species include disease resistance, sterility, and enhanced growth. Despite these notable advances, off-target effect and non-clarification of trait-related genes among other technical challenges hinder full realization of CRISPR/Cas potentials in fish breeding. In addition, current regulatory and risk assessment frameworks are not fit-for purpose regarding the challenges of CRISPR/Cas notwithstanding that public and regulatory acceptance are key to commercialization of products of the new technology. In this study, we discuss how CRISPR/Cas can be used to overcome some of these limitations focusing on diseases and environmental release in farmed fish aquaculture. We further present technical limitations, regulatory and risk assessment challenges of the use of CRISPR/Cas, and proffer research strategies that will provide much-needed data for regulatory decisions, risk assessments, increased public awareness and sustainable applications of CRISPR/Cas in fish aquaculture with emphasis on Atlantic salmon (Salmo salar) breeding.},
}

@article {pmid34303184,
year = {2021},
author = {Shi, S and Qi, N and Nielsen, J},
title = {Microbial production of chemicals driven by CRISPR-Cas systems.},
journal = {Current opinion in biotechnology},
volume = {73},
number = {},
pages = {34-42},
doi = {10.1016/j.copbio.2021.07.002},
pmid = {34303184},
issn = {1879-0429},
abstract = {Microorganisms have provided an attractive route for biosynthesis of various chemicals from renewable resources. CRISPR-Cas systems have served as powerful mechanisms for generating cell factories with desirable properties by manipulating nucleic acids quickly and efficiently. The CRISPR-Cas system provides a toolbox with excellent opportunities for identifying better biocatalysts, multiplexed fine-tuning of metabolic flux, efficient utilization of low-cost substrates, and improvement of metabolic robustness. The overall goal of this review highlights recent advances in the development of microbial cell factories for chemical production using various CRISPR-Cas systems. The perspectives for further development or applications of CRISPR-Cas systems for strain improvement are also discussed.},
}

@article {pmid34302285,
year = {2021},
author = {Qu, J and Xie, Y and Guo, Z and Liu, X and Jiang, J and Chen, T and Li, K and Hu, Z and Luo, D},
title = {Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {34302285},
issn = {1559-0305},
support = {2018JJ1021//Hunan Province Science Fund for Distinguished Young Scholars/ ; 2017SK2172//the Key R&D Program of Hunan Province/ ; 2017JJ2003//the Natural Science Foundation of Hunan Province/ ; 2017JJ2004//the Natural Science Foundation of Hunan Province/ ; 2017SK51101//the Hunan Province Technological Innovation Guiding Plan, Clinical Medical Technology Innovation Guidance Project/ ; B2017182, B20180378, B20180266//the Health Department project of the Hunan Province/ ; jsyf2017021, jsyf2017023 and zdyf201838)//the Science and Technique Foundation of Chenzhou/ ; },
abstract = {Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5' phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3' of the target RNA which is recognized by 5' sequence of the gDNA. These results confirmed that NgAgo-gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3' of the target RNA, which is a new finding that has not been reported in the past.},
}

@article {pmid34302019,
year = {2021},
author = {Li, CY and Steighner, JR and Sweatt, G and Thiele, TR and Juntti, SA},
title = {Manipulation of the Tyrosinase gene permits improved CRISPR/Cas editing and neural imaging in cichlid fish.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {15138},
pmid = {34302019},
issn = {2045-2322},
support = {RGY0079/2018//Human Frontier Science Program/ ; RGY0079/2018//Human Frontier Science Program/ ; },
abstract = {Direct tests of gene function have historically been performed in a limited number of model organisms. The CRISPR/Cas system is species-agnostic, offering the ability to manipulate genes in a range of models, enabling insights into evolution, development, and physiology. Astatotilapia burtoni, a cichlid fish from the rivers and shoreline around Lake Tanganyika, has been extensively studied in the laboratory to understand evolution and the neural control of behavior. Here we develop protocols for the creation of CRISPR-edited cichlids and create a broadly useful mutant line. By manipulating the Tyrosinase gene, which is necessary for eumelanin pigment production, we describe a fast and reliable approach to quantify and optimize gene editing efficiency. Tyrosinase mutants also remove a major obstruction to imaging, enabling visualization of subdermal structures and fluorophores in situ. These protocols will facilitate broad application of CRISPR/Cas9 to studies of cichlids as well as other non-traditional model aquatic species.},
}

@article {pmid34298471,
year = {2021},
author = {Bonini, A and Poma, N and Vivaldi, F and Biagini, D and Bottai, D and Tavanti, A and Di Francesco, F},
title = {A label-free impedance biosensing assay based on CRISPR/Cas12a collateral activity for bacterial DNA detection.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {204},
number = {},
pages = {114268},
doi = {10.1016/j.jpba.2021.114268},
pmid = {34298471},
issn = {1873-264X},
abstract = {The rapid and selective identification in the clinical setting of pathogenic bacteria causing healthcare associated infections (HAIs) and in particular blood stream infections (BSIs) is a major challenge, as the number of people affected worldwide and the associated mortality are on the rise. In fact, traditional laboratory techniques such culture and polymerase chain reaction (PCR)-based methodologies are often associated to long turnaround times, which justify the pressing need for the development of rapid, specific and portable point of care devices. The recently discovered clustered regularly interspaced short palindromic repeat loci (CRISPR) and the new class of programmable endonuclease enzymes called CRISPR associated proteins (Cas) have revolutionised molecular diagnostics. The use of Cas proteins in optical and electrochemical biosensing devices has significantly improved the detection of nucleic acids in clinical samples. In this study, a CRISPR/Cas12a system was coupled with electrochemical impedance spectroscopy (EIS) measurements to develop a label-free biosensing assay for the detection of Escherichia coli and Staphylococcus aureus, two bacterial species commonly associated to BSI infections. The programmable Cas12a endonuclease activity, induced by a specific guide RNA (gRNA), and the triggered collateral activity were assessed in in vitro restriction analyses, and evaluated thanks to impedance measurements using a modified gold electrode. The Cas12a/gRNA system was able to specifically recognize amplicons from different clinical isolates of E. coli and S. aureus with a limit of detection of 3 nM and a short turnaround time approximately of 1.5 h. To the best of our knowledge, this is the first biosensing device based on CRISPR/Cas12a label free impedance assay.},
}

@article {pmid34297925,
year = {2021},
author = {Bosch, B and DeJesus, MA and Poulton, NC and Zhang, W and Engelhart, CA and Zaveri, A and Lavalette, S and Ruecker, N and Trujillo, C and Wallach, JB and Li, S and Ehrt, S and Chait, BT and Schnappinger, D and Rock, JM},
title = {Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2021.06.033},
pmid = {34297925},
issn = {1097-4172},
abstract = {Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.},
}

@article {pmid34294050,
year = {2021},
author = {Straume, AH and Kjærner-Semb, E and Skaftnesmo, KO and Güralp, H and Lillico, S and Wargelius, A and Edvardsen, RB},
title = {Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors.},
journal = {BMC genomics},
volume = {22},
number = {1},
pages = {563},
pmid = {34294050},
issn = {1471-2164},
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Nucleotides ; Oligonucleotides ; *Salmo salar/genetics ; },
abstract = {BACKGROUND: New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon.

RESULTS: Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively).

CONCLUSIONS: In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.},
}

Alleles
Animals
*CRISPR-Cas Systems/genetics
Gene Editing
Nucleotides
Oligonucleotides
*Salmo salar/genetics

@article {pmid34281288,
year = {2021},
author = {Koster, C and van den Hurk, KT and Lewallen, CF and Talib, M and Ten Brink, JB and Boon, CJF and Bergen, AA},
title = {The Lrat-/- Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34281288},
issn = {1422-0067},
support = {UZ 2016 - 21//Stichting Uitzicht including the Stichting MD Nederland, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Retina Fonds Nederland, Oogfonds/ ; B20160043//Rotterdamse Stichting Blindenbelangen/ ; //Stichting Blindenhulp/ ; //Stichting voor Ooglijders/ ; 42//Stichting Lijf en Leven/ ; },
mesh = {Acyltransferases/*deficiency/*genetics ; Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Disease Models, Animal ; Electroretinography ; Female ; Gene Knockout Techniques ; Humans ; Male ; Mice ; Ophthalmoscopy ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Transgenic ; Retinitis Pigmentosa/diagnostic imaging/*genetics/physiopathology ; Sequence Deletion ; Tomography, Optical Coherence ; Vision, Ocular ; },
abstract = {PURPOSE: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle.

METHODS: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat-/- and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays.

RESULTS: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat-/- animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat-/- animals. Lrat-/- animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat-/- animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals.

CONCLUSIONS: Our Lrat-/- rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.},
}

Acyltransferases/*deficiency/*genetics
Animals
Behavior, Animal
CRISPR-Cas Systems
Disease Models, Animal
Electroretinography
Female
Gene Knockout Techniques
Humans
Male
Mice
Ophthalmoscopy
RNA, Messenger/genetics/metabolism
Rats
Rats, Transgenic
Retinitis Pigmentosa/diagnostic imaging/*genetics/physiopathology
Sequence Deletion
Tomography, Optical Coherence
Vision, Ocular

@article {pmid34230471,
year = {2021},
author = {Eggers, EJ and van der Burgt, A and van Heusden, SAW and de Vries, ME and Visser, RGF and Bachem, CWB and Lindhout, P},
title = {Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4141},
pmid = {34230471},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Chromosome Mapping ; Chromosomes, Plant ; Diploidy ; Genes, Plant/*genetics ; Genotype ; Heterozygote ; Magnoliopsida ; *Plant Breeding ; Plant Proteins/*genetics ; Pollen/genetics ; Seeds/metabolism ; Self-Incompatibility in Flowering Plants/genetics ; Solanum tuberosum/*genetics ; },
abstract = {Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.},
}

CRISPR-Cas Systems
Chromosome Mapping
Chromosomes, Plant
Diploidy
Genes, Plant/*genetics
Genotype
Heterozygote
Magnoliopsida
*Plant Breeding
Plant Proteins/*genetics
Pollen/genetics
Seeds/metabolism
Self-Incompatibility in Flowering Plants/genetics
Solanum tuberosum/*genetics

@article {pmid34209672,
year = {2021},
author = {Liang, Y and Biswas, S and Kim, B and Bailey-Serres, J and Septiningsih, EM},
title = {Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34209672},
issn = {1422-0067},
support = {2017-67013-26194//U.S. Department of Agriculture/ ; },
mesh = {CRISPR-Cas Systems ; Calcium-Binding Proteins/genetics ; Gene Editing ; *Genes, Plant ; Oryza/*physiology ; Phenotype ; Plant Development/genetics ; Plants, Genetically Modified ; *Regeneration ; Reproducibility of Results ; Seeds/genetics/growth & development ; *Transformation, Genetic ; },
abstract = {Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.},
}

CRISPR-Cas Systems
Calcium-Binding Proteins/genetics
Gene Editing
*Genes, Plant
Oryza/*physiology
Phenotype
Plant Development/genetics
Plants, Genetically Modified
*Regeneration
Reproducibility of Results
Seeds/genetics/growth & development
*Transformation, Genetic

@article {pmid34201772,
year = {2021},
author = {DeLeo, KR and Baral, SS and Houser, A and James, A and Sewell, P and Pandey, S and DiMario, PJ},
title = {Drosophila to Explore Nucleolar Stress.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34201772},
issn = {1422-0067},
support = {MCB 1712975//National Science Foundation/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Nucleolus/*genetics ; Coiled Bodies/genetics/*pathology ; Drosophila Proteins/antagonists & inhibitors/*genetics ; Drosophila melanogaster/*genetics/physiology ; *Gene Expression Regulation, Developmental ; Larva/genetics/growth & development ; Phosphoproteins ; RNA-Binding Proteins/antagonists & inhibitors/*genetics ; Ribosomes/genetics/metabolism ; *Stress, Physiological ; },
abstract = {Nucleolar stress occurs when ribosome production or function declines. Nucleolar stress in stem cells or progenitor cells often leads to disease states called ribosomopathies. Drosophila offers a robust system to explore how nucleolar stress causes cell cycle arrest, apoptosis, or autophagy depending on the cell type. We provide an overview of nucleolar stress in Drosophila by depleting nucleolar phosphoprotein of 140 kDa (Nopp140), a ribosome biogenesis factor (RBF) in nucleoli and Cajal bodies (CBs). The depletion of Nopp140 in eye imaginal disc cells generates eye deformities reminiscent of craniofacial deformities associated with the Treacher Collins syndrome (TCS), a human ribosomopathy. We show the activation of c-Jun N-terminal Kinase (JNK) in Drosophila larvae homozygous for a Nopp140 gene deletion. JNK is known to induce the expression of the pro-apoptotic Hid protein and autophagy factors Atg1, Atg18.1, and Atg8a; thus, JNK is a central regulator in Drosophila nucleolar stress. Ribosome abundance declines upon Nopp140 loss, but unusual cytoplasmic granules accumulate that resemble Processing (P) bodies based on marker proteins, Decapping Protein 1 (DCP1) and Maternal expression at 31B (Me31B). Wild type brain neuroblasts (NBs) express copious amounts of endogenous coilin, but coilin levels decline upon nucleolar stress in most NB types relative to the Mushroom body (MB) NBs. MB NBs exhibit resilience against nucleolar stress as they maintain normal coilin, Deadpan, and EdU labeling levels.},
}

Animals
CRISPR-Cas Systems
Cell Nucleolus/*genetics
Coiled Bodies/genetics/*pathology
Drosophila Proteins/antagonists & inhibitors/*genetics
Drosophila melanogaster/*genetics/physiology
*Gene Expression Regulation, Developmental
Larva/genetics/growth & development
Phosphoproteins
RNA-Binding Proteins/antagonists & inhibitors/*genetics
Ribosomes/genetics/metabolism
*Stress, Physiological

@article {pmid34200006,
year = {2021},
author = {Zhao, Y and Zou, J and Gao, Q and Xie, S and Cao, J and Zhou, H},
title = {CMAS and ST3GAL4 Play an Important Role in the Adsorption of Influenza Virus by Affecting the Synthesis of Sialic Acid Receptors.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34200006},
issn = {1422-0067},
support = {32025036, 31772752 and 31761133005//National Natural Science Foundation of China/ ; 2016YFD0500205//National Key Research and Development Program of China/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Influenza A virus/*physiology ; N-Acetylneuraminic Acid/metabolism ; N-Acylneuraminate Cytidylyltransferase/*antagonists & inhibitors/genetics/metabolism ; Orthomyxoviridae Infections/metabolism/pathology/*virology ; Receptors, Cell Surface/*metabolism ; Sialyltransferases/*antagonists & inhibitors ; Swine ; *Virus Replication ; },
abstract = {Influenza A viruses (IAVs) initiate infection by attaching Hemagglutinin (HA) on the viral envelope to sialic acid (SA) receptors on the cell surface. Importantly, HA of human IAVs has a higher affinity for α-2,6-linked SA receptors, and avian strains prefer α-2,3-linked SA receptors, whereas swine strains have a strong affinity for both SA receptors. Host gene CMAS and ST3GAL4 were found to be essential for IAV attachment and entry. Loss of CMAS and ST3GAL4 hindered the synthesis of sialic acid receptors, which in turn prevented the adsorption of IAV. Further, the knockout of CMAS had an effect on the adsorption of swine, avian and human IAVs. However, ST3GAL4 knockout prevented the adsorption of swine and avian IAV and the impact on avian IAV was more distinct, whereas it had no effect on the adsorption of human IAV. Collectively, our findings demonstrate that knocking out CMAS and ST3GAL4 negatively regulated IAV replication by inhibiting the synthesis of SA receptors, which also provides new insights into the production of gene-edited animals in the future.},
}

Animals
CRISPR-Cas Systems
Influenza A virus/*physiology
N-Acetylneuraminic Acid/metabolism
N-Acylneuraminate Cytidylyltransferase/*antagonists & inhibitors/genetics/metabolism
Orthomyxoviridae Infections/metabolism/pathology/*virology
Receptors, Cell Surface/*metabolism
Sialyltransferases/*antagonists & inhibitors
Swine
*Virus Replication

@article {pmid34199901,
year = {2021},
author = {Horodecka, K and Düchler, M},
title = {CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34199901},
issn = {1422-0067},
support = {UMO-2019/35/B/NZ7/03256//Narodowym Centrum Nauki/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Extracellular Vesicles/*genetics ; *Gene Editing ; *Gene Transfer Techniques ; Genetic Therapy/*methods ; Humans ; },
abstract = {The establishment of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology for eukaryotic gene editing opened up new avenues not only for the analysis of gene function but also for therapeutic interventions. While the original methodology allowed for targeted gene disruption, recent technological advancements yielded a rich assortment of tools to modify genes and gene expression in various ways. Currently, clinical applications of this technology fell short of expectations mainly due to problems with the efficient and safe delivery of CRISPR/Cas9 components to living organisms. The targeted in vivo delivery of therapeutic nucleic acids and proteins remain technically challenging and further limitations emerge, for instance, by unwanted off-target effects, immune reactions, toxicity, or rapid degradation of the transfer vehicles. One approach that might overcome many of these limitations employs extracellular vesicles as intercellular delivery devices. In this review, we first introduce the CRISPR/Cas9 system and its latest advancements, outline major applications, and summarize the current state of the art technology using exosomes or microvesicles for transporting CRISPR/Cas9 constituents into eukaryotic cells.},
}

Animals
*CRISPR-Cas Systems
Extracellular Vesicles/*genetics
*Gene Editing
*Gene Transfer Techniques
Genetic Therapy/*methods
Humans

@article {pmid34106048,
year = {2021},
author = {Kumar, M and Gulati, S and Ansari, AH and Phutela, R and Acharya, S and Azhar, M and Murthy, J and Kathpalia, P and Kanakan, A and Maurya, R and Vasudevan, JS and S, A and Pandey, R and Maiti, S and Chakraborty, D},
title = {FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34106048},
issn = {2050-084X},
support = {Graduate student fellowship//University Grants Commission/ ; CLP-0033//IUSSTF/ ; HCP0008//CSIR Sickle Cell Anemia Mission/ ; SSP 2001//Tata Steel/ ; GAP0198//Lady Tata Young Investigator/ ; Graduate Student fellowship//CSIR/ ; Research Associateship//CSIR/ ; Graduate Student fellowship//Indian Council of Medical Research/ ; MLP 2005//CSIR/ ; CLP-0031//Fondation Botnar/ ; CLP-0034//Intel Corporation/ ; HCP0008//CSIR/ ; GAP0198//Lady Tata Memorial Trust/ ; },
mesh = {COVID-19/*genetics ; *COVID-19 Nucleic Acid Testing ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; SARS-CoV-2/*genetics ; },
abstract = {The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.},
}

COVID-19/*genetics
*COVID-19 Nucleic Acid Testing
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
SARS-CoV-2/*genetics

@article {pmid33773911,
year = {2021},
author = {Ferreira, TR and Couñago, RM and Moretti, NS},
title = {Raising the Bar(-seq) in Leishmania Genetic Screens.},
journal = {Trends in parasitology},
volume = {37},
number = {5},
pages = {367-369},
doi = {10.1016/j.pt.2021.03.002},
pmid = {33773911},
issn = {1471-5007},
mesh = {Animals ; CRISPR-Cas Systems ; *Leishmania/genetics ; Life Cycle Stages ; },
abstract = {Our understanding of regulatory factors in Leishmania differentiation has long been restricted by the available genetic tools, but the availability of CRISPR/Cas9 has changed the landscape forever. Recently, Baker and Catta-Preta et al. applied Cas9 editing and kinome-wide bar-seq to dissect the function of 204 kinases in the Leishmania mexicana life cycle.},
}

Animals
CRISPR-Cas Systems
*Leishmania/genetics
Life Cycle Stages

@article {pmid33759566,
year = {2021},
author = {Hirshorn, ST and Steele, N and Zavros, Y},
title = {Modeling pancreatic pathophysiology using genome editing of adult stem cell-derived and induced pluripotent stem cell (iPSC)-derived organoids.},
journal = {American journal of physiology. Gastrointestinal and liver physiology},
volume = {320},
number = {6},
pages = {G1142-G1150},
doi = {10.1152/ajpgi.00329.2020},
pmid = {33759566},
issn = {1522-1547},
support = {5U19AI11649105//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01DK083402-10//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; P30 CA023074/CA/NCI NIH HHS/United States ; },
mesh = {Adult Stem Cells/*cytology ; Animals ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Organoids/*cytology ; Pancreas/*cytology ; },
abstract = {In recent years, organoids have become a novel in vitro method to study gastrointestinal organ development, physiology, and disease. An organoid, in short, may be defined as a miniaturized organ that can be grown from adult stem cells in vitro and studied at the microscopic level. Organoids have been used in multitudes of different ways to study the physiology of different human diseases including gastrointestinal cancers such as pancreatic cancer. The development of genome editing based on the bacterial defense mechanism clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a laboratory tool that provides the opportunity to study the effects of specific genetic changes on organ development, physiology, and disease. The CRISPR/Cas9 approach can be combined with organoid technology including the use of induced pluripotent stem cell (iPSC)-derived and tissue-derived organoids. The goal of this review is to provide highlights on the development of organoid technology, and the use of this culture system to study the pathophysiology of specific mutations in the development of pancreatic and gastric cancers.NEW & NOTEWORTHY The goal of this review is not only to provide highlights on the development of organoid technology but also to subsequently use this information to study the pathophysiology of those specific mutations in the formation of malignant pancreatic and gastric cancer.},
}

Adult Stem Cells/*cytology
Animals
CRISPR-Cas Systems
*Gene Editing
Humans
Induced Pluripotent Stem Cells/*cytology
Organoids/*cytology
Pancreas/*cytology

@article {pmid33672015,
year = {2021},
author = {Emmanouilidis, I and Fili, N and Cook, AW and Hari-Gupta, Y and Dos Santos, Á and Wang, L and Martin-Fernandez, ML and Ellis, PJI and Toseland, CP},
title = {A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.},
journal = {Biomolecules},
volume = {11},
number = {2},
pages = {},
pmid = {33672015},
issn = {2218-273X},
support = {MR/M020606/1/MRC_/Medical Research Council/United Kingdom ; 19130001//Central Laser Facility, Science and Technology Facilities Council/ ; },
mesh = {*CRISPR-Cas Systems ; Cell Survival ; Cisplatin/pharmacology ; Computer Simulation ; *DNA Breaks, Double-Stranded ; *DNA Damage ; DNA Repair ; Electroporation ; Endonucleases/genetics ; Escherichia coli/metabolism ; Gene Editing/methods ; Genome, Human ; Genomic Instability ; Genomics ; Humans ; Microscopy, Confocal ; Microscopy, Fluorescence ; Mutagens ; RNA, Guide ; Stochastic Processes ; },
abstract = {Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.},
}

*CRISPR-Cas Systems
Cell Survival
Cisplatin/pharmacology
Computer Simulation
*DNA Breaks, Double-Stranded
*DNA Damage
DNA Repair
Electroporation
Endonucleases/genetics
Escherichia coli/metabolism
Gene Editing/methods
Genome, Human
Genomic Instability
Genomics
Humans
Microscopy, Confocal
Microscopy, Fluorescence
Mutagens
RNA, Guide
Stochastic Processes

@article {pmid33671263,
year = {2021},
author = {Seroussi, E},
title = {Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing.},
journal = {Genes},
volume = {12},
number = {2},
pages = {},
pmid = {33671263},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/genetics ; DNA Copy Number Variations/*genetics ; Gene Dosage/*genetics ; INDEL Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA Editing/genetics ; Sequence Analysis, DNA/*trends ; Software ; },
abstract = {Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.},
}

CRISPR-Cas Systems/genetics
DNA Copy Number Variations/*genetics
Gene Dosage/*genetics
INDEL Mutation/genetics
Polymorphism, Single Nucleotide/genetics
RNA Editing/genetics
Sequence Analysis, DNA/*trends
Software

@article {pmid33621063,
year = {2021},
author = {Rabiee, N and Bagherzadeh, M and Heidarian Haris, M and Ghadiri, AM and Matloubi Moghaddam, F and Fatahi, Y and Dinarvand, R and Jarahiyan, A and Ahmadi, S and Shokouhimehr, M},
title = {Polymer-Coated NH2-UiO-66 for the Codelivery of DOX/pCRISPR.},
journal = {ACS applied materials & interfaces},
volume = {13},
number = {9},
pages = {10796-10811},
doi = {10.1021/acsami.1c01460},
pmid = {33621063},
issn = {1944-8252},
mesh = {Acrylic Resins/*chemistry/toxicity ; Adsorption ; *CRISPR-Cas Systems ; Doxorubicin/chemistry/*metabolism ; Drug Carriers/*chemistry/toxicity ; Drug Liberation ; Gene Transfer Techniques ; Green Chemistry Technology ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Metal-Organic Frameworks/*chemistry/toxicity ; Polyhydroxyethyl Methacrylate/*chemistry/toxicity ; Porosity ; },
abstract = {Herein, the NH2-UiO-66 metal organic framework (MOF) has been green synthesized with the assistance of high gravity to provide a suitable and safe platform for drug loading. The NH2-UiO-66 MOF was characterized using a field-emission scanning electron microscope, transmission electron microscope (TEM), X-ray diffraction, and zeta potential analysis. Doxorubicin was then encapsulated physically on the porosity of the green MOF. Two different stimulus polymers, p(HEMA) and p(NIPAM), were used as the coating agents of the MOFs. Doxorubicin was loaded onto the polymer-coated MOFs as well, and a drug payload of more than 51% was obtained, which is a record by itself. In the next step, pCRISPR was successfully tagged on the surface of the modified MOFs, and the performance of the final nanosystems were evaluated by the GFP expression. In addition, successful loadings and internalizations of doxorubicin were investigated via confocal laser scanning microscopy. Cellular images from the HeLa cell line for the UiO-66@DOX@pCRISPR and GMA-UiO-66@DOX@pCRISPR do not show any promising and successful gene transfections, with a maximum EGFP of 1.6%; however, the results for the p(HEMA)-GMA-UiO-66@DOX@pCRISPR show up to 4.3% transfection efficiency. Also, the results for the p(NIPAM)-GMA-UiO-66@DOX@pCRISPR showed up to 6.4% transfection efficiency, which is the first and superior report of a MOF-based nanocarrier for the delivery of pCRISPR. Furthermore, the MTT assay does not shown any critical cytotoxicity, which is a promising result for further biomedical applications. At the end of the study, the morphologies of all of the nanomaterials were screened after drug and gene delivery procedures and showed partial degradation of the nanomaterial. However, the cubic structure of the MOFs has been shown in TEM, and this is further proof of the stability of these green MOFs for biomedical applications.},
}

Acrylic Resins/*chemistry/toxicity
Adsorption
*CRISPR-Cas Systems
Doxorubicin/chemistry/*metabolism
Drug Carriers/*chemistry/toxicity
Drug Liberation
Gene Transfer Techniques
Green Chemistry Technology
Green Fluorescent Proteins/genetics
HEK293 Cells
HeLa Cells
Humans
Metal-Organic Frameworks/*chemistry/toxicity
Polyhydroxyethyl Methacrylate/*chemistry/toxicity
Porosity

@article {pmid33591941,
year = {2021},
author = {Chen, F and Fang, S and Du, Y and Ghosh, A and Reed, MN and Long, Y and Suppiramaniam, V and Tang, S and Hong, H},
title = {CRISPR/Cas9-mediated CysLT1R deletion reverses synaptic failure, amyloidosis and cognitive impairment in APP/PS1 mice.},
journal = {Aging},
volume = {13},
number = {5},
pages = {6634-6661},
pmid = {33591941},
issn = {1945-4589},
mesh = {Amyloid beta-Protein Precursor ; Amyloidosis/*prevention & control ; Animals ; *CRISPR-Cas Systems ; Cognitive Dysfunction/*prevention & control ; Disease Models, Animal ; *Gene Deletion ; Mice, Knockout ; Mice, Transgenic ; Neuronal Plasticity ; Receptors, Leukotriene/*genetics ; *Synaptic Transmission ; },
abstract = {As a major pathological hallmark of Alzheimer's disease (AD), amyloid-β (Aβ) is regarded as a causative factor for cognitive impairment. Extensive studies have found Aβ induces a series of pathophysiological responses, finally leading to memory loss in AD. Our previous results demonstrated that cysteinyl leukotrienes receptor 1 (CysLT1R) antagonists improved exogenous Aβ-induced memory impairment. But the role of CysLT1R in AD and its underlying mechanisms still remain elusive. In this study, we investigated CysLT1R levels in AD patients and APP/PS1 mice. We also generated APP/PS1-CysLT1R-/- mice by clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated CysLT1R deletion in APP/PS1 mice and studied the effect of CysLT1R knockout on amyloidogenesis, synapse structure and plasticity, cognition, neuroinflammation, and kynurenine pathway. These attributes were also studied after lentivirus-mediated knockdown of CysLT1R gene in APP/PS1 mice. We found that CysLT1R knockout or knockdown could conserve synaptic structure and plasticity, and improve cognition in APP/PS1 mice. These effects were associated with concurrent decreases in amyloid processing, reduced neuroinflammation and suppression of the kynurenine pathway. Our study demonstrates that CysLT1R deficiency can mediate several beneficial effects against AD pathogenesis, and genetic/pharmacological ablation of this protein could be a potential therapeutic option for AD.},
}

Amyloid beta-Protein Precursor
Amyloidosis/*prevention & control
Animals
*CRISPR-Cas Systems
Cognitive Dysfunction/*prevention & control
Disease Models, Animal
*Gene Deletion
Mice, Knockout
Mice, Transgenic
Neuronal Plasticity
Receptors, Leukotriene/*genetics
*Synaptic Transmission

@article {pmid33396657,
year = {2020},
author = {Mukae, T and Okumura, S and Watanobe, T and Yoshii, K and Tagami, T and Oishi, I},
title = {Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens.},
journal = {Genes},
volume = {12},
number = {1},
pages = {},
pmid = {33396657},
issn = {2073-4425},
mesh = {Animals ; Animals, Genetically Modified ; Antibodies, Monoclonal/*biosynthesis/isolation & purification ; Bioreactors ; *CRISPR-Cas Systems ; Chickens/*genetics ; Egg White/*chemistry ; Female ; Gene Editing/methods ; Humans ; Plasmids/chemistry/metabolism ; Receptor, ErbB-2/*antagonists & inhibitors/genetics/immunology ; Trastuzumab/biosynthesis/isolation & purification ; Zygote/chemistry/metabolism ; },
abstract = {Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4-1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.},
}

Animals
Animals, Genetically Modified
Antibodies, Monoclonal/*biosynthesis/isolation & purification
Bioreactors
*CRISPR-Cas Systems
Chickens/*genetics
Egg White/*chemistry
Female
Gene Editing/methods
Humans
Plasmids/chemistry/metabolism
Receptor, ErbB-2/*antagonists & inhibitors/genetics/immunology
Trastuzumab/biosynthesis/isolation & purification
Zygote/chemistry/metabolism

@article {pmid33396568,
year = {2020},
author = {Ben Shlush, I and Samach, A and Melamed-Bessudo, C and Ben-Tov, D and Dahan-Meir, T and Filler-Hayut, S and Levy, AA},
title = {CRISPR/Cas9 Induced Somatic Recombination at the CRTISO Locus in Tomato.},
journal = {Genes},
volume = {12},
number = {1},
pages = {},
pmid = {33396568},
issn = {2073-4425},
mesh = {Alleles ; Biological Assay ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Crosses, Genetic ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA, Plant/chemistry/*genetics/metabolism ; Gene Editing/methods ; Genetic Loci ; *Genome, Plant ; Lycopersicon esculentum/*genetics/metabolism ; Plant Breeding/methods ; *Recombinational DNA Repair ; },
abstract = {Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.},
}

Alleles
Biological Assay
CRISPR-Cas Systems
Carotenoids/metabolism
Crosses, Genetic
DNA Breaks, Double-Stranded
*DNA End-Joining Repair
DNA, Plant/chemistry/*genetics/metabolism
Gene Editing/methods
Genetic Loci
*Genome, Plant
Lycopersicon esculentum/*genetics/metabolism
Plant Breeding/methods
*Recombinational DNA Repair

@article {pmid33298142,
year = {2020},
author = {Yang, Y and Tang, T and Feng, B and Li, S and Hou, N and Ma, X and Jiang, L and Xin, X and Chen, Q},
title = {Disruption of Plasmodium falciparum histidine-rich protein 2 may affect haem metabolism in the blood stage.},
journal = {Parasites & vectors},
volume = {13},
number = {1},
pages = {611},
pmid = {33298142},
issn = {1756-3305},
support = {2018ZX09738-005//Ministry of Science and Technology of the People's Republic of China/ ; },
mesh = {Animals ; Antigens, Protozoan/*genetics/*metabolism ; Antimalarials/metabolism ; Base Sequence ; Binding Sites ; CRISPR-Cas Systems ; Diagnostic Tests, Routine ; Gene Targeting ; Heme/*metabolism ; Hemoglobins ; Humans ; Life Cycle Stages ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/genetics/*metabolism ; Polymerase Chain Reaction ; Protozoan Proteins/*genetics/*metabolism ; Transcriptome ; },
abstract = {BACKGROUND: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages.

METHODS: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2--3D7 parasites.

RESULTS: Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1).

CONCLUSION: Our data revealed changes in the transcriptome expression profile of the Pfhrp2--3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.},
}

Animals
Antigens, Protozoan/*genetics/*metabolism
Antimalarials/metabolism
Base Sequence
Binding Sites
CRISPR-Cas Systems
Diagnostic Tests, Routine
Gene Targeting
Heme/*metabolism
Hemoglobins
Humans
Life Cycle Stages
Malaria, Falciparum/parasitology
Plasmodium falciparum/genetics/*metabolism
Polymerase Chain Reaction
Protozoan Proteins/*genetics/*metabolism
Transcriptome

@article {pmid33167309,
year = {2020},
author = {Su, L and Shi, C and Huang, X and Wang, Y and Li, G},
title = {Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing.},
journal = {Genes},
volume = {11},
number = {11},
pages = {},
pmid = {33167309},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/physiology ; Endonucleases/genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Genotype ; Lancelets/*genetics ; Phenotype ; Phylogeny ; },
abstract = {The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.},
}

Animals
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/physiology
Endonucleases/genetics
Gene Editing/*methods
Genome, Plant/genetics
Genotype
Lancelets/*genetics
Phenotype
Phylogeny

@article {pmid33107387,
year = {2021},
author = {Man, JCK and van Duijvenboden, K and Krijger, PHL and Hooijkaas, IB and van der Made, I and de Gier-de Vries, C and Wakker, V and Creemers, EE and de Laat, W and Boukens, BJ and Christoffels, VM},
title = {Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart.},
journal = {Circulation research},
volume = {128},
number = {1},
pages = {115-129},
doi = {10.1161/CIRCRESAHA.120.317045},
pmid = {33107387},
issn = {1524-4571},
mesh = {Animals ; Atrial Natriuretic Factor/*genetics/metabolism ; Binding Sites ; Binding, Competitive ; CRISPR-Cas Systems ; Cell Line ; Disease Models, Animal ; *Enhancer Elements, Genetic ; Epigenesis, Genetic ; Gene Expression Regulation, Developmental ; Humans ; Hypertrophy, Left Ventricular/*genetics/metabolism/pathology ; Mice, Knockout ; *Multigene Family ; Myocardial Infarction/*genetics/metabolism/pathology ; Myocytes, Cardiac/*metabolism/pathology ; Natriuretic Peptide, Brain/*genetics/metabolism ; Promoter Regions, Genetic ; },
abstract = {RATIONALE: ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), encoded by the clustered genes Nppa and Nppb, are important prognostic, diagnostic, and therapeutic proteins in cardiac disease. The spatiotemporal expression pattern and stress-induction of the Nppa and Nppb are tightly regulated, possibly involving their coregulation by an evolutionary conserved enhancer cluster.

OBJECTIVE: To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlying Nppa-Nppb coregulation in vivo.

METHODS AND RESULTS: By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream of Nppb. Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof, Nppb and flanking regions or the entire genomic block spanning Nppa-Nppb, respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic, and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression of Nppa and Nppb but not of any other gene. Enhancer cluster-deficient mice showed enlarged hearts before and after birth, similar to Nppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters of Nppa and Nppb in a competitive rather than a cooperative mode, resulting in increased Nppa expression when Nppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity when its target genes are absent. In enhancer cluster-deficient animals, Nppa was induced but remained low in the postmyocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal to Nppa.

CONCLUSIONS: Coordinated ventricular expression of Nppa and Nppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy.},
}

Animals
Atrial Natriuretic Factor/*genetics/metabolism
Binding Sites
Binding, Competitive
CRISPR-Cas Systems
Cell Line
Disease Models, Animal
*Enhancer Elements, Genetic
Epigenesis, Genetic
Gene Expression Regulation, Developmental
Humans
Hypertrophy, Left Ventricular/*genetics/metabolism/pathology
Mice, Knockout
*Multigene Family
Myocardial Infarction/*genetics/metabolism/pathology
Myocytes, Cardiac/*metabolism/pathology
Natriuretic Peptide, Brain/*genetics/metabolism
Promoter Regions, Genetic

@article {pmid32035013,
year = {2020},
author = {Lin, MH and Pope, BD and Sasaki, T and Keeley, DP and Sherwood, DR and Miner, JH},
title = {Mammalian hemicentin 1 is assembled into tracks in the extracellular matrix of multiple tissues.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {249},
number = {6},
pages = {775-788},
pmid = {32035013},
issn = {1097-0177},
support = {R01 DK078314/DK/NIDDK NIH HHS/United States ; R35 GM118049/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Basement Membrane/metabolism ; CRISPR-Cas Systems/genetics ; Extracellular Matrix/*metabolism ; Extracellular Matrix Proteins/genetics/*metabolism ; Hair Follicle/cytology/metabolism ; Kidney/metabolism ; Mice ; Mice, Knockout ; Sclera/cytology/metabolism ; },
abstract = {BACKGROUND: Hemicentins (HMCNs) are a family of extracellular matrix proteins first identified in Caenorhabditis elegans, with two orthologs (HMCN1 and 2) in vertebrates. In worms, HMCN is deposited at specific sites where it forms long, fine tracks that link two tissues by connecting adjacent basement membranes (BMs). By generating CRISPR/Cas9-mediated Hmcn1 and Hmcn2 knockout mice, we tested the hypothesis that HMCNs perform similar functions in mammals.

RESULTS: Hmcn1 -/- mice were viable and fertile. Using new, knockout mouse-validated HMCN1 antibodies, HMCN1 was detected in wild-type mice as fine tracks along the BM of hair and whisker follicles, in the sclera of the eyes, and in the lumen of some lymphoid conduits. It was also observed in the mesangial matrix of the kidney glomerulus. However, HMCN1 deficiency did not affect the functions of these tissues, including adherence of coat hairs and whiskers, the sieving function of lymphoid conduits, or the immune response to injected antigens. HMCN2 deficiency did not lead to any discernible phenotypes on its own or when combined with HMCN1 deficiency.

CONCLUSION: That Hmcn1 -/- , Hmcn2 -/- , and Hmcn1/2 double knockout mice did not display any overt phenotypes implicates compensation by other members of the fibulin family.},
}

Animals
Basement Membrane/metabolism
CRISPR-Cas Systems/genetics
Extracellular Matrix/*metabolism
Extracellular Matrix Proteins/genetics/*metabolism
Hair Follicle/cytology/metabolism
Kidney/metabolism
Mice
Mice, Knockout
Sclera/cytology/metabolism

@article {pmid31765060,
year = {2020},
author = {Rodríguez Cruz, PM and Cossins, J and Cheung, J and Maxwell, S and Jayawant, S and Herbst, R and Waithe, D and Kornev, AP and Palace, J and Beeson, D},
title = {Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure.},
journal = {Human mutation},
volume = {41},
number = {3},
pages = {619-631},
pmid = {31765060},
issn = {1098-1004},
support = {P 31199/FWF_/Austrian Science Fund FWF/Austria ; MR/M006824/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adrenergic beta-2 Receptor Agonists/pharmacology/therapeutic use ; Alleles ; Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Cell Line ; DNA Mutational Analysis ; Female ; Gene Targeting ; Humans ; Mice ; Models, Molecular ; Molecular Conformation ; Muscle Proteins/metabolism ; *Mutation ; Myasthenic Syndromes, Congenital/*diagnosis/drug therapy/*genetics/metabolism ; Pedigree ; Phosphorylation ; Receptor Protein-Tyrosine Kinases/chemistry/*genetics/metabolism ; Receptors, Cholinergic/chemistry/*genetics/metabolism ; Structure-Activity Relationship ; Synapses/*genetics/metabolism ; },
abstract = {MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with β2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.},
}

Adrenergic beta-2 Receptor Agonists/pharmacology/therapeutic use
Alleles
Amino Acid Substitution
Animals
CRISPR-Cas Systems
Cell Line
DNA Mutational Analysis
Female
Gene Targeting
Humans
Mice
Models, Molecular
Molecular Conformation
Muscle Proteins/metabolism
*Mutation
Myasthenic Syndromes, Congenital/*diagnosis/drug therapy/*genetics/metabolism
Pedigree
Phosphorylation
Receptor Protein-Tyrosine Kinases/chemistry/*genetics/metabolism
Receptors, Cholinergic/chemistry/*genetics/metabolism
Structure-Activity Relationship
Synapses/*genetics/metabolism

@article {pmid34284989,
year = {2021},
author = {Wang, HT and Li, TT and Huang, X and Ma, RL and Liu, QY},
title = {Application of genetic modification technologies in molecular design breeding of sheep.},
journal = {Yi chuan = Hereditas},
volume = {43},
number = {6},
pages = {580-600},
doi = {10.16288/j.yczz.21-087},
pmid = {34284989},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Plant Breeding ; Sheep/genetics ; Technology ; },
abstract = {Genetic modification technologies can be used for modifying animal genome to express exogenous genes or affect the function of endogenous genes. In animal breeding, genetic modification technologies allow the rapid generation of germplasms with beneficial traits. It includes traditional genetic modification, virus or sperm carrier-mediated genetic modification and nuclease-mediated genome editing, especially the CRISPR/Cas9, one of the artificial nuclease genome editing technologies, have been applied in genome editing in many domestic animals including sheep (Ovis aries). Compared with conventional strategies used for animal breeding, there is great value for sheep breeding improvement by using genome editing technology, which is more effective and timesaving. In this review, we summarize the approaches of genetic modification in sheep and discuss the possibility of molecular design and breeding of sheep by genome editing technologies. We also identify the potential bottlenecks and challenges of these technologies in sheep breeding.},
}

Animals
*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Plant Breeding
Sheep/genetics
Technology

@article {pmid34284985,
year = {2021},
author = {Jiang, J and Zhao, AQ and Xie, T and Chen, SW and Li, JS},
title = {Construction of genome-wide protein tagging cell and mouse libraries.},
journal = {Yi chuan = Hereditas},
volume = {43},
number = {7},
pages = {704-714},
doi = {10.16288/j.yczz.21-163},
pmid = {34284985},
issn = {0253-9772},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome ; Male ; Mice ; },
abstract = {Mice are the most widely used model organism for the study of gene functions and disease mechanisms through the generation of gene-modified mice. Since the 1980s, different genetic manipulation technologies have been developed to reveal gene functions in vivo, including homologous recombination strategies mediated by embryonic stem cells, transgenic strategies mediated by gametes, and the latest genetic modification strategies based on CRISPR/Cas9 technology. Semi-cloning technology mediated by "artificial spermatids" (androgenetic haploid embryonic stem cells, also termed sperm-like stem cells) is developed by Chinese scientists in 2012. In combination with CRISPR/Cas9, semi-cloning technology enables one-step generation of gene-modified mice through injection of "artificial spermatids" with specific gene modifications into oocytes. It has the characteristics of short construction cycle, high efficiency, low cost, and high application compatibility. In 2017, the Center for Excellence in Molecular Cell Science (CEMCS) of CAS has launched the genome tagging project (GTP) based on "artificial spermatid"-mediated semi-cloning technology. The ambitious goal of GTP is to tag every protein in mice and construct a unique mouse library that maintains the genome-wide protein-tagging mouse models. Subsequently, the GTP center was established at CEMCS to pursue the project. GTP center developed strategies to generate protein-tagging cells and mice. Briefly, a tag sequence is precisely inserted in a specific protein- coding gene endogenously in cultured "artificial spermatids"in vitro to build a cell library, in which, each cell line carrying a specific protein tag. The tagged cells could be further used as a sperm replacement to produce tagged mice in one step upon injection into oocytes. The tagged mouse library enables global analysis of protein expression, localization, and complexes using standard tag-based assays in vivo. By April 2021, the GTP center has generated 1532 tagged cell lines, 277 of which have been successfully used to produce tagged mice through oocyte injection. A total of 242 tagged mouse strains have been distributed to 66 research teams in 32 research institutions of 15 districts in 3 countries. The database of tagging product resources has been established and released regularly on the GTP website for scientists to inquire and order. Later, more information about GTP products, such as mouse breeding, protein tissue expression map, published literature, etc., will also be successively published on the GTP website. The GTP center will provide a standardized platform for protein function research, which may dramatically promote the development of life science and clinical transformation.},
}

Animals
Animals, Genetically Modified
*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Genome
Male
Mice

@article {pmid34210975,
year = {2021},
author = {Yang, L and Chan, AKN and Miyashita, K and Delaney, CD and Wang, X and Li, H and Pokharel, SP and Li, S and Li, M and Xu, X and Lu, W and Liu, Q and Mattson, N and Chen, KY and Wang, J and Yuan, YC and Horne, D and Rosen, ST and Soto-Feliciano, Y and Feng, Z and Hoshii, T and Xiao, G and Müschen, M and Chen, J and Armstrong, SA and Chen, CW},
title = {High-resolution characterization of gene function using single-cell CRISPR tiling screen.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4063},
pmid = {34210975},
issn = {2041-1723},
support = {P50 CA206963/CA/NCI NIH HHS/United States ; K99 CA197498/CA/NCI NIH HHS/United States ; R00 CA197498/CA/NCI NIH HHS/United States ; R01 CA176745/CA/NCI NIH HHS/United States ; R37 CA233691/CA/NCI NIH HHS/United States ; P30 CA033572/CA/NCI NIH HHS/United States ; R01 CA236626/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drug Screening Assays, Antitumor ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genetic Testing ; Genome, Human ; Histone-Lysine N-Methyltransferase/chemistry/genetics ; Histones ; Humans ; Models, Molecular ; Mutagenesis ; Neoplasms/*genetics ; *Phenotype ; Transcriptome ; },
abstract = {Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Here, we present "sc-Tiling," which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Drug Screening Assays, Antitumor
Gene Editing
Gene Expression Regulation, Neoplastic
Genetic Testing
Genome, Human
Histone-Lysine N-Methyltransferase/chemistry/genetics
Histones
Humans
Models, Molecular
Mutagenesis
Neoplasms/*genetics
*Phenotype
Transcriptome

@article {pmid34119838,
year = {2021},
author = {Yang, Y and Liu, J and Zhou, X},
title = {A CRISPR-based and post-amplification coupled SARS-CoV-2 detection with a portable evanescent wave biosensor.},
journal = {Biosensors & bioelectronics},
volume = {190},
number = {},
pages = {113418},
doi = {10.1016/j.bios.2021.113418},
pmid = {34119838},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The continuing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), has spread globally and its reliable diagnosis is one of the foremost priorities for protecting public health. Herein a rapid (<1 h), easy-to-implement, and accurate CRISPR-based evanescent wave fluorescence biosensing platform for detection of SARS-CoV-2 is reported. The collateral effect of Cas13a is combined with a universal autonomous enzyme-free hybridization chain reaction (HCR) by designing a cleavage hairpin reporter, which is cleaved upon target recognition, and hence releasing the initiator sequence to trigger the downstream HCR circuits. Detection of HCR assemblies is accomplished by first adsorbing to the desthiobiotin-modified optical fiber, followed by fluorescence emission induced by an evanescent field. Three Cas13a crRNAs targeting the genes of S, N and Orf1ab of SARS-CoV-2 are programmed to specifically target SARS-CoV-2 or broadly detect related coronavirus strains, such as MERS-CoV and SARS-CoV. The HCR amplification coupled Cas13a-based biosensing platform is capable of rapid detection of SARS-CoV-2 with attomolar sensitivity. This method is further validated by adding target RNA of SARS-CoV-2 in negative oropharyngeal swabs. The good discrimination capability of this technique demonstrates its promising potential for point-of-care diagnosis of COVID-19.},
}

*Biosensing Techniques
*COVID-19
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Nucleic Acid Amplification Techniques
RNA, Viral
SARS-CoV-2
Sensitivity and Specificity

@article {pmid33974980,
year = {2021},
author = {Süntar, I and Çetinkaya, S and Haydaroğlu, ÜS and Habtemariam, S},
title = {Bioproduction process of natural products and biopharmaceuticals: Biotechnological aspects.},
journal = {Biotechnology advances},
volume = {50},
number = {},
pages = {107768},
doi = {10.1016/j.biotechadv.2021.107768},
pmid = {33974980},
issn = {1873-1899},
mesh = {*Biological Products ; Biotechnology ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
abstract = {Decades of research have been put in place for developing sustainable routes of bioproduction of high commercial value natural products (NPs) on the global market. In the last few years alone, we have witnessed significant advances in the biotechnological production of NPs. The development of new methodologies has resulted in a better understanding of the metabolic flux within the organisms, which have driven manipulations to improve production of the target product. This was further realised due to the recent advances in the omics technologies such as genomics, transcriptomics, proteomics, metabolomics and secretomics, as well as systems and synthetic biology. Additionally, the combined application of novel engineering strategies has made possible avenues for enhancing the yield of these products in an efficient and economical way. Invention of high-throughput technologies such as next generation sequencing (NGS) and toolkits for genome editing Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) have been the game changers and provided unprecedented opportunities to generate rationally designed synthetic circuits which can produce complex molecules. This review covers recent advances in the engineering of various hosts for the production of bioactive NPs and biopharmaceuticals. It also highlights general approaches and strategies to improve their biosynthesis with higher yields in a perspective of plants and microbes (bacteria, yeast and filamentous fungi). Although there are numerous reviews covering this topic on a selected species at a time, our approach herein is to give a comprehensive understanding about state-of-art technologies in different platforms of organisms.},
}

*Biological Products
Biotechnology
*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing

@article {pmid33766595,
year = {2021},
author = {Pan, S and Su, Y and Sun, B and Hao, R and Gao, X and Han, B},
title = {Knockout of CD147 inhibits the proliferation, invasion, and drug resistance of human oral cancer CAL27 cells in Vitro and in Vivo.},
journal = {International journal of biological macromolecules},
volume = {181},
number = {},
pages = {378-389},
doi = {10.1016/j.ijbiomac.2021.03.102},
pmid = {33766595},
issn = {1879-0003},
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Apoptosis/drug effects ; Basigin/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Disease Models, Animal ; Doxorubicin/pharmacology/therapeutic use ; *Drug Resistance, Neoplasm/drug effects ; *Gene Knockout Techniques ; Humans ; Matrix Metalloproteinases/metabolism ; Mice, Nude ; Mouth Neoplasms/*drug therapy/*pathology ; Neoplasm Invasiveness ; },
abstract = {With the development of modern biomedicine, research on the molecular mechanism of tumors has developed gradually. The CD147 gene has been applied to tumor molecular targeted therapy, and significant differences were found in the expression of the CD147 gene in different tumor tissues and normal tissues. Many previous studies have also shown that the expression of the CD147 gene plays a crucial role in the development of tumors. To understand whether CD147 can be used as a therapeutic target for oral cancer, CRISPR/Cas9 gene-editing technology was used to knock out the CD147 gene in cal27 cells to obtain knockout cell lines. Using CCK-8, Transwell, RT-PCR, and Western blotting, the proliferation and invasion abilities of the knockout cell lines were decreased significantly, and the expression of matrix metalloproteinase was also inhibited. Next, a subcutaneously transplanted tumor model in nude mice was constructed to detect the effect of the CD147 gene on tumors. Subcutaneous tumor growth and immunohistochemistry results showed that the proliferation and doxorubicin resistance of knockout cell line were significantly inhibited compared with those in the wild-type group. These results indicated that knocking out CD147 significantly reduced the proliferation and invasion of cal27 cells, and CD147 may be a potential therapeutic target for oral cancer.},
}

Animals
Antineoplastic Agents/pharmacology/therapeutic use
Apoptosis/drug effects
Basigin/*metabolism
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Movement/drug effects
Cell Proliferation/drug effects
Disease Models, Animal
Doxorubicin/pharmacology/therapeutic use
*Drug Resistance, Neoplasm/drug effects
*Gene Knockout Techniques
Humans
Matrix Metalloproteinases/metabolism
Mice, Nude
Mouth Neoplasms/*drug therapy/*pathology
Neoplasm Invasiveness

@article {pmid33689075,
year = {2021},
author = {Singina, GN and Sergiev, PV and Lopukhov, AV and Rubtsova, MP and Taradajnic, NP and Ravin, NV and Shedova, EN and Taradajnic, TE and Polejaeva, IA and Dozev, AV and Brem, G and Dontsova, OA and Zinovieva, NA},
title = {Production of a Cloned Offspring and CRISPR/Cas9 Genome Editing of Embryonic Fibroblasts in Cattle.},
journal = {Doklady. Biochemistry and biophysics},
volume = {496},
number = {1},
pages = {48-51},
pmid = {33689075},
issn = {1608-3091},
mesh = {Animals ; Animals, Genetically Modified/embryology/*genetics ; *CRISPR-Cas Systems ; Cattle/embryology/*genetics ; Cloning, Organism/*methods ; Embryo, Mammalian/*cytology/metabolism ; Fibroblasts/*cytology/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Nuclear Transfer Techniques ; },
abstract = {Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.},
}

Animals
Animals, Genetically Modified/embryology/*genetics
*CRISPR-Cas Systems
Cattle/embryology/*genetics
Cloning, Organism/*methods
Embryo, Mammalian/*cytology/metabolism
Fibroblasts/*cytology/metabolism
Gene Editing/*methods
Gene Knockout Techniques/methods
Nuclear Transfer Techniques

@article {pmid33572577,
year = {2021},
author = {Majchrzak-Celińska, A and Warych, A and Szoszkiewicz, M},
title = {Novel Approaches to Epigenetic Therapies: From Drug Combinations to Epigenetic Editing.},
journal = {Genes},
volume = {12},
number = {2},
pages = {},
pmid = {33572577},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Methylation/genetics ; Drug Combinations ; Epigenesis, Genetic/*genetics ; Epigenome/*genetics ; Gene Editing/trends ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {Cancer development involves both genetic and epigenetic alterations. Aberrant epigenetic modifications are reversible, allowing excellent opportunities for therapeutic intervention. Nowadays, several epigenetic drugs are used worldwide to treat, e.g., myelodysplastic syndromes and leukemias. However, overcoming resistance and widening the therapeutic profiles are the most important challenges faced by traditional epigenetic drugs. Recently, novel approaches to epigenetic therapies have been proposed. Next-generation epigenetic drugs, with longer half-life and better bioavailability, are being developed and tested. Since epigenetic phenomena are interdependent, treatment modalities include co-administration of two different epigenetic drugs. In order to sensitize cancer cells to chemotherapy, epigenetic drugs are administered prior to chemotherapy, or both epigenetic drug and chemotherapy are used together to achieve synergistic effects and maximize treatment efficacy. The combinations of epigenetic drug with immunotherapy are being tested, because they have proved to enhance antitumor immune responses. The next approach involves targeting the metabolic causes of epigenetic changes, i.e., enzymes which, when mutated, produce oncometabolites. Finally, epigenome editing makes it possible to modify individual chromatin marks at a defined region with unprecedented specificity and efficiency. This review summarizes the above attempts in fulfilling the promise of epigenetic drugs in the effective cancer treatment.},
}

CRISPR-Cas Systems/*genetics
DNA Methylation/genetics
Drug Combinations
Epigenesis, Genetic/*genetics
Epigenome/*genetics
Gene Editing/trends
Humans
Neoplasms/genetics/*therapy

@article {pmid33322084,
year = {2020},
author = {Smith, RH and Chen, YC and Seifuddin, F and Hupalo, D and Alba, C and Reger, R and Tian, X and Araki, D and Dalgard, CL and Childs, RW and Pirooznia, M and Larochelle, A},
title = {Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones.},
journal = {Genes},
volume = {11},
number = {12},
pages = {},
pmid = {33322084},
issn = {2073-4425},
support = {Z99 HL999999/HL/NHLBI NIH HHS/United States ; },
mesh = {Adult ; *CRISPR-Cas Systems ; Chromosomes, Human, Pair 19/*genetics ; Chromosomes, Human, Pair 2/*genetics ; *Clone Cells ; Female ; *Gene Editing ; Genetic Loci ; *Hematopoietic Stem Cells ; Humans ; Receptors, CXCR4/genetics ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.},
}

Adult
*CRISPR-Cas Systems
Chromosomes, Human, Pair 19/*genetics
Chromosomes, Human, Pair 2/*genetics
*Clone Cells
Female
*Gene Editing
Genetic Loci
*Hematopoietic Stem Cells
Humans
Receptors, CXCR4/genetics

@article {pmid32687866,
year = {2020},
author = {Southern, SJ and Oyston, PCF},
title = {Genome editing of Francisella tularensis using (CRISPR-Cas9).},
journal = {Journal of microbiological methods},
volume = {176},
number = {},
pages = {106004},
doi = {10.1016/j.mimet.2020.106004},
pmid = {32687866},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems ; Francisella tularensis/*genetics ; Gene Editing/*methods ; *Genome, Bacterial ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system is a powerful tool for gene editing in eukaryotic genomes but is still being developed for editing bacterial genomes. Here we describe the construction of an all-in-one vector for generating potentially scarless deletion mutants in Francisella tularensis LVS using a CRISPR-Cas9-based system.},
}

*CRISPR-Cas Systems
Francisella tularensis/*genetics
Gene Editing/*methods
*Genome, Bacterial

@article {pmid32070859,
year = {2020},
author = {Zuo, Z and Liu, J},
title = {Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage.},
journal = {Current opinion in structural biology},
volume = {62},
number = {},
pages = {166-174},
pmid = {32070859},
issn = {1879-033X},
support = {R15 HL147265/HL/NHLBI NIH HHS/United States ; },
mesh = {Allosteric Regulation ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; DNA/*metabolism ; DNA Cleavage ; Humans ; Protein Engineering/*methods ; RNA, Guide/*metabolism ; },
abstract = {The CRISPR-Cas9 system from Streptococcus pyogenes has been exploited as a programmable RNA-guided DNA-targeting and DNA-editing platform. This evolutionary tool enables diverse genetic manipulations with unprecedented precision and ease. Cas9 is an allosteric enzyme, which is allosterically regulated in conformational activation, target recognition, and DNA cleavage. Here, we outline the underlying allosteric control over the Cas9 complex assembly and targeting specificity. We further review the strategies for mitigating intrinsic Cas9 off-target effects through allosteric modulations and the advances in engineering controllable Cas9 systems that are responsive to external allosteric signals. Future development of highly specific, tunable CRISPR-Cas9 systems through allosteric modulations would greatly benefit applications that require both conditional control and high precision.},
}

Allosteric Regulation
CRISPR-Associated Protein 9/*metabolism
*CRISPR-Cas Systems
DNA/*metabolism
DNA Cleavage
Humans
Protein Engineering/*methods
RNA, Guide/*metabolism

@article {pmid34289313,
year = {2021},
author = {Cameranesi, MM and Kurth, D and Repizo, GD},
title = {Acinetobacter defence mechanims against biological aggressors and their use as alternative therapeutic applications.},
journal = {Critical reviews in microbiology},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/1040841X.2021.1939266},
pmid = {34289313},
issn = {1549-7828},
abstract = {Several Acinetobacter strains are important nosocomial pathogens, with Acinetobacter baumannii being the species of greatest worldwide concern due to its multi-drug resistance and the recent appearance of hyper-virulent strains in the clinical setting. Colonisation of this environment is associated with a multitude of bacterial factors, and the molecular features that promote environmental persistence in abiotic surfaces, including intrinsic desiccation resistance, biofilm formation and motility, have been previously addressed. On the contrary, mechanisms enabling Acinetobacter spp. survival when faced against other biological competitors are starting to be characterised. Among them, secretion systems (SS) of different types, such as the T5bSS (Contact-dependent inhibition systems) and the T6SS, confer adaptive advantages against bacterial aggressors. Regarding mechanisms of defence against bacteriophages, such as toxin-antitoxin, restriction-modification, Crispr-Cas and CBASS, among others, have been identified but remain poorly characterised. In view of this, we aimed to summarise the present knowledge on defence mechanisms that enable niche establishment in members of the Acinetobacter genus. Different proposals are also described for the use of some components of these systems as molecular tools to treat Acinetobacter infections.},
}

@article {pmid34288688,
year = {2021},
author = {Ahmad, S and Tang, L and Shahzad, R and Mawia, AM and Rao, GS and Jamil, S and Wei, C and Sheng, Z and Shao, G and Wei, X and Hu, P and Mahfouz, MM and Hu, S and Tang, S},
title = {CRISPR-Based Crop Improvements: A Way Forward to Achieve Zero Hunger.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.1c02653},
pmid = {34288688},
issn = {1520-5118},
abstract = {Zero hunger is one of the sustainable development goals set by the United Nations in 2015 to achieve global food security by 2030. The current harvest of crops is insufficient; feeding the world's population and meeting the goal of zero hunger by 2030 will require larger and more consistent crop production. Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) technology is widely used for the plant genome editing. In this review, we consider this technology as a potential tool for achieving zero hunger. We provide a comprehensive overview of CRISPR-Cas technology and its most important applications for food crops' improvement. We also conferred current and potential technological breakthroughs that will help in breeding future crops to end global hunger. The regulatory aspects of deploying this technology in commercial sectors, bioethics, and the production of transgene-free plants are also discussed. We hope that the CRISPR-Cas system will accelerate the breeding of improved crop cultivars compared with conventional breeding and pave the way toward the zero hunger goal.},
}

@article {pmid34287045,
year = {2021},
author = {Wang, Q and Liu, Y and Han, C and Yang, M and Huang, F and Duan, X and Wang, S and Yu, Y and Liu, J and Yang, H and Lu, D and Zhao, H and Zhang, Y and Qin, Q},
title = {Efficient RNA virus targeting via CRISPR-CasRx in fish.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {JVI0046121},
doi = {10.1128/JVI.00461-21},
pmid = {34287045},
issn = {1098-5514},
abstract = {The emergence of the CRISPR-Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR-Cas13 system has been used to target RNA. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. Importance RNA viruses are most important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find a widely effective ways to inhibit RNA viruses. Therefore, we urgently need to develop effective antiviral methods. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a non-enveloped positive-strand RNA virus, is one of the most serious viral pathogens infecting more than 40 cultured fish species resulting in huge economic losses worldwide. Here, we establish a novel efective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioices) as model. Our data show that CasRx have the most robust for RNA virus interference applications in fish and demonstrate its suitability for studying key questions relating to virus biology.},
}

@article {pmid34286726,
year = {2021},
author = {Xie, R and Wang, Y and Gong, S},
title = {External stimuli-responsive nanoparticles for spatially and temporally controlled delivery of CRISPR-Cas genome editors.},
journal = {Biomaterials science},
volume = {},
number = {},
pages = {},
doi = {10.1039/d1bm00558h},
pmid = {34286726},
issn = {2047-4849},
abstract = {The CRISPR-Cas9 system is a powerful tool for genome editing, which can potentially lead to new therapies for genetic diseases. To date, various viral and non-viral delivery systems have been developed for the delivery of CRISPR-Cas9 in vivo. However, spatially and temporally controlled genome editing is needed to enhance the specificity in organs/tissues and minimize the off-target effects of editing. In this review, we summarize the state-of-the-art non-viral vectors that exploit external stimuli (i.e., light, magnetic field, and ultrasound) for spatially and temporally controlled genome editing and their in vitro and in vivo applications.},
}

@article {pmid34286046,
year = {2018},
author = {Mohanraju, P and Van Der Oost, J and Jinek, M and Swarts, DC},
title = {Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1.},
journal = {Bio-protocol},
volume = {8},
number = {9},
pages = {e2842},
doi = {10.21769/BioProtoc.2842},
pmid = {34286046},
issn = {2331-8325},
abstract = {This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments. Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli.},
}

@article {pmid34285780,
year = {2021},
author = {Tay, AP and Hosking, B and Hosking, C and Bauer, DC and Wilson, LOW},
title = {INSIDER: alignment-free detection of foreign DNA sequences.},
journal = {Computational and structural biotechnology journal},
volume = {19},
number = {},
pages = {3810-3816},
doi = {10.1016/j.csbj.2021.06.045},
pmid = {34285780},
issn = {2001-0370},
abstract = {External DNA sequences can be inserted into an organism's genome either through natural processes such as gene transfer, or through targeted genome engineering strategies. Being able to robustly identify such foreign DNA is a crucial capability for health and biosecurity applications, such as anti-microbial resistance (AMR) detection or monitoring gene drives. This capability does not exist for poorly characterised host genomes or with limited information about the integrated sequence. To address this, we developed the INserted Sequence Information DEtectoR (INSIDER). INSIDER analyses whole genome sequencing data and identifies segments of potentially foreign origin by their significant shift in k-mer signatures. We demonstrate the power of INSIDER to separate integrated DNA sequences from normal genomic sequences on a synthetic dataset simulating the insertion of a CRISPR-Cas gene drive into wild-type yeast. As a proof-of-concept, we use INSIDER to detect the exact AMR plasmid in whole genome sequencing data from a Citrobacter freundii patient isolate. INSIDER streamlines the process of identifying integrated DNA in poorly characterised wild species or when the insert is of unknown origin, thus enhancing the monitoring of emerging biosecurity threats.},
}

@article {pmid34284709,
year = {2021},
author = {Sherstyuk, VV and Zakian, SM},
title = {Generation of Transgenic Rat Embryonic Stem Cells Using the CRISPR/Cpf1 System for Inducible Gene Knockout.},
journal = {Biochemistry. Biokhimiia},
volume = {86},
number = {7},
pages = {843-851},
doi = {10.1134/S0006297921070051},
pmid = {34284709},
issn = {1608-3040},
abstract = {Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism. To study functions of the essential genes, as well as the processes of cell differentiation, the method of induced knockout is widely used. The CreERT2/loxP system allows obtaining an inducible knockout in cells expressing tamoxifen-inducible Cre recombinase (CreERT2) and containing loxP sites flanking the target gene by adding 4-hydroxy tamoxifen to the culture medium. However, the rat ESC lines expressing CreERT2 are absent. In this work, we tested three CRISPR/Cas systems for introduction of double-strand breaks into the Rosa26 locus in the rat ESCs and inserted tamoxifen-dependent Cre recombinase into this locus using the CRISPR/Cpf1 system. It was shown that the obtained transgenic rat ESC lines retained the characteristics of pluripotent cells. Tamoxifen-inducible Cre recombinase activity was analyzed using a reporter vector.},
}

@article {pmid34017121,
year = {2021},
author = {Cheung, P and Schaffert, S and Chang, SE and Dvorak, M and Donato, M and Macaubas, C and Foecke, MH and Li, TM and Zhang, L and Coan, JP and Schulert, GS and Grom, AA and Henderson, LA and Nigrovic, PA and Elias, JE and Gozani, O and Mellins, ED and Khatri, P and Utz, PJ and Kuo, AJ},
title = {Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation.},
journal = {Nature immunology},
volume = {22},
number = {6},
pages = {711-722},
pmid = {34017121},
issn = {1529-2916},
support = {K08 AR073339/AR/NIAMS NIH HHS/United States ; T32 CA009302/CA/NCI NIH HHS/United States ; R01 AI125197/AI/NIAID NIH HHS/United States ; P30 AR070253/AR/NIAMS NIH HHS/United States ; R01 AR059049/AR/NIAMS NIH HHS/United States ; U19 AI110491/AI/NIAID NIH HHS/United States ; P30 AR070549/AR/NIAMS NIH HHS/United States ; K08 AR072075/AR/NIAMS NIH HHS/United States ; R01 AR073201/AR/NIAMS NIH HHS/United States ; R35 GM139569/GM/NIGMS NIH HHS/United States ; U19 AI109662/AI/NIAID NIH HHS/United States ; U19 AI057229/AI/NIAID NIH HHS/United States ; R01 AR061297/AR/NIAMS NIH HHS/United States ; },
mesh = {Adolescent ; Arthritis, Juvenile/blood/genetics/*immunology ; CRISPR-Cas Systems/genetics ; Cathepsin G/genetics/metabolism ; Cell Differentiation/genetics/*immunology ; Cell Nucleus/metabolism ; Child ; Child, Preschool ; Chromatin/metabolism ; Enzyme Assays ; Epigenesis, Genetic/*immunology ; Epigenomics ; Female ; Gene Knockout Techniques ; Histones/*metabolism ; Humans ; Jurkat Cells ; Leukocyte Elastase/genetics/metabolism ; Leukocytes, Mononuclear/immunology/*metabolism ; Macrophages/*immunology/metabolism ; Male ; Myeloblastin/genetics/metabolism ; Primary Cell Culture ; Proteolysis ; RNA-Seq ; Recombinant Proteins/genetics/metabolism ; THP-1 Cells ; Young Adult ; },
abstract = {Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.},
}

Adolescent
Arthritis, Juvenile/blood/genetics/*immunology
CRISPR-Cas Systems/genetics
Cathepsin G/genetics/metabolism
Cell Differentiation/genetics/*immunology
Cell Nucleus/metabolism
Child
Child, Preschool
Chromatin/metabolism
Enzyme Assays
Epigenesis, Genetic/*immunology
Epigenomics
Female
Gene Knockout Techniques
Histones/*metabolism
Humans
Jurkat Cells
Leukocyte Elastase/genetics/metabolism
Leukocytes, Mononuclear/immunology/*metabolism
Macrophages/*immunology/metabolism
Male
Myeloblastin/genetics/metabolism
Primary Cell Culture
Proteolysis
RNA-Seq
Recombinant Proteins/genetics/metabolism
THP-1 Cells
Young Adult

@article {pmid33713445,
year = {2021},
author = {Xu, W and Cheng, H and Zhu, S and Cheng, J and Ji, H and Zhang, B and Cao, S and Wang, C and Tong, G and Zhen, C and Mu, L and Zhou, Y and Cheng, Y},
title = {Functional understanding of secondary cell wall cellulose synthases in Populus trichocarpa via the Cas9/gRNA-induced gene knockouts.},
journal = {The New phytologist},
volume = {231},
number = {4},
pages = {1478-1495},
doi = {10.1111/nph.17338},
pmid = {33713445},
issn = {1469-8137},
mesh = {CRISPR-Cas Systems ; Cell Wall/metabolism ; Cellulose/metabolism ; Gene Knockout Techniques ; Glucosyltransferases ; *Populus/genetics/metabolism ; RNA, Guide ; Wood/metabolism ; },
abstract = {Plant cellulose is synthesized by a large plasma membrane-localized cellulose synthase (CesA) complex. However, an overall functional determination of secondary cell wall (SCW) CesAs is still lacking in trees, especially one based on gene knockouts. Here, the Cas9/gRNA-induced knockouts of PtrCesA4, 7A, 7B, 8A and 8B genes were produced in Populus trichocarpa. Based on anatomical, immunohistochemical and wood composition evidence, we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level. Complete loss of PtrCesA4, 7A/B or 8A/B led to similar morphological abnormalities, indicating similar and nonredundant genetic functions. The absence of the gelatinous (G) layer, one-layer-walled fibres and a 90% decrease in cellulose in these mutant woods revealed that the three classes of SCW PtrCesAs are essential for multilayered SCW structure and wood G-fibre. In addition, the mutant primary and secondary phloem fibres lost the n(G + L)- and G-layers and retained the thicker S-layers (L, lignified; S, secondary). Together with polysaccharide immunolocalization data, these findings suggest differences in the role of SCW PtrCesAs-synthesized cellulose in wood and phloem fibre wall structures. Overall, this functional understanding of the SCW PtrCesAs provides further insights into the impact of lacking cellulose biosynthesis on growth, SCW, wood G-fibre and phloem fibre wall structures in the tree.},
}

CRISPR-Cas Systems
Cell Wall/metabolism
Cellulose/metabolism
Gene Knockout Techniques
Glucosyltransferases
*Populus/genetics/metabolism
RNA, Guide
Wood/metabolism

@article {pmid33318646,
year = {2021},
author = {Berber, B and Aydin, C and Kocabas, F and Guney-Esken, G and Yilancioglu, K and Karadag-Alpaslan, M and Caliseki, M and Yuce, M and Demir, S and Tastan, C},
title = {Gene editing and RNAi approaches for COVID-19 diagnostics and therapeutics.},
journal = {Gene therapy},
volume = {28},
number = {6},
pages = {290-305},
pmid = {33318646},
issn = {1476-5462},
mesh = {*COVID-19/diagnosis/therapy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Oligonucleotides, Antisense ; *RNA Interference ; },
abstract = {The novel coronavirus pneumonia (COVID-19) is a highly infectious acute respiratory disease caused by Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV-2) (Prec Clin Med 2020;3:9-13, Lancet 2020;395:497-506, N. Engl J Med 2020a;382:1199-207, Nature 2020;579:270-3). SARS-CoV-2 surveillance is essential to controlling widespread transmission. However, there are several challenges associated with the diagnostic of the COVID-19 during the current outbreak (Liu and Li (2019), Nature 2020;579:265-9, N. Engl J Med 2020;382:727-33). Firstly, the high number of cases overwhelms diagnostic test capacity and proposes the need for a rapid solution for sample processing (Science 2018;360:444-8). Secondly, SARS-CoV-2 is closely related to other important coronavirus species and subspecies, so detection assays can give false-positive results if they are not efficiently specific to SARS-CoV-2. Thirdly, patients with suspected SARS-CoV-2 infection sometimes have a different respiratory viral infection or co-infections with SARS-CoV-2 and other respiratory viruses (MedRxiv 2020a;1-18). Confirmation of the COVID-19 is performed mainly by virus isolation followed by RT-PCR and sequencing (N. Engl J Med 2020;382:727-33, MedRxiv 2020a, Turkish J Biol 2020;44:192-202). The emergence and outbreak of the novel coronavirus highlighted the urgent need for new therapeutic technologies that are fast, precise, stable, easy to manufacture, and target-specific for surveillance and treatment. Molecular biology tools that include gene-editing approaches such as CRISPR-Cas12/13-based SHERLOCK, DETECTR, CARVER and PAC-MAN, antisense oligonucleotides, antisense peptide nucleic acids, ribozymes, aptamers, and RNAi silencing approaches produced with cutting-edge scientific advances compared to conventional diagnostic or treatment methods could be vital in COVID-19 and other future outbreaks. Thus, in this review, we will discuss potent the molecular biology approaches that can revolutionize diagnostic of viral infections and therapies to fight COVID-19 in a highly specific, stable, and efficient way.},
}

*COVID-19/diagnosis/therapy
CRISPR-Cas Systems
*Gene Editing
Humans
Oligonucleotides, Antisense
*RNA Interference

@article {pmid33256757,
year = {2020},
author = {Song, I and Kim, J and Baek, K and Choi, Y and Shin, B and Jin, E},
title = {The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {220},
pmid = {33256757},
issn = {1475-2859},
support = {20190066//Ministry of Oceans and Fisheries (KR)/ ; 2014M1A8A1 049273//Ministry of Science, ICT and Future Planning/ ; NRF-2020R1A2C2011998//Ministry of Science, ICT and Future Planning/ ; },
mesh = {Algal Proteins/genetics ; Biosynthetic Pathways ; CRISPR-Cas Systems ; Chlamydomonas reinhardtii/*genetics/*metabolism ; Gene Knockout Techniques ; Industrial Microbiology ; Intramolecular Lyases/*genetics ; Metabolic Engineering ; Oxidoreductases/genetics ; Zeaxanthins/*biosynthesis ; },
abstract = {BACKGROUND: Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin.

RESULTS: In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of α-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24 mg L- 1) and content (7.28 mg g- 1) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84 mg L- 1.

CONCLUSIONS: A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.},
}

Algal Proteins/genetics
Biosynthetic Pathways
CRISPR-Cas Systems
Chlamydomonas reinhardtii/*genetics/*metabolism
Gene Knockout Techniques
Industrial Microbiology
Intramolecular Lyases/*genetics
Metabolic Engineering
Oxidoreductases/genetics
Zeaxanthins/*biosynthesis

@article {pmid32749114,
year = {2020},
author = {Noor, S and Rasul, A and Iqbal, MS and Ahmed, B and Akash, MSH and Qadir, MI},
title = {Inhibition of Hepatitis B Virus with the Help of CRISPR/Cas9 Technology.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {30},
number = {3},
pages = {273-278},
doi = {10.1615/CritRevEukaryotGeneExpr.2020028453},
pmid = {32749114},
issn = {1045-4403},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Circular/metabolism ; DNA, Viral/metabolism ; Gene Editing ; Genetic Engineering ; Genetic Therapy ; Genome, Viral ; Hepatitis B/*therapy ; Hepatitis B virus/*genetics/physiology ; Humans ; Mutagenesis, Site-Directed ; *Virus Replication ; },
abstract = {Hepatitis B infection caused by hepatitis B virus (HBV) is a serious health issue worldwide. Existing therapeutic strategies hardly eradicate HBV infections, and they fail to attain complete cure. Advanced treatment strategies are urgently needed to successfully terminate further spread of HBV infection and eliminate hidden reservoirs of virus. Recently, a novel RNA-guided gene editing tool, known as the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system, has been established. It facilitates site-specific mutagenesis and reveals a new way to develop applicable techniques for disease treatment, such as extermination of infectious agents like HBV This study highlights the current developments in CRISPR/Cas9 technology and its importance for target-specific inhibition of HBV genome. Benefits, challenges, feasible solutions, and proposed guidelines for forthcoming study in CRISPR/Cas9 are described to highlight the possible cures of and treatments for chronic HBV infection.},
}

Animals
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA, Circular/metabolism
DNA, Viral/metabolism
Gene Editing
Genetic Engineering
Genetic Therapy
Genome, Viral
Hepatitis B/*therapy
Hepatitis B virus/*genetics/physiology
Humans
Mutagenesis, Site-Directed
*Virus Replication

@article {pmid32227559,
year = {2020},
author = {Lamas-Toranzo, I and Martínez-Moro, A and O Callaghan, E and Millán-Blanca, G and Sánchez, JM and Lonergan, P and Bermejo-Álvarez, P},
title = {RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos.},
journal = {Molecular reproduction and development},
volume = {87},
number = {5},
pages = {542-549},
pmid = {32227559},
issn = {1098-2795},
mesh = {Animals ; Animals, Genetically Modified ; Benzamides/*pharmacology ; CRISPR-Cas Systems/*drug effects/physiology ; Cattle/*embryology ; Cells, Cultured ; DNA Breaks, Double-Stranded/drug effects ; DNA End-Joining Repair/*drug effects ; Embryo Culture Techniques ; Embryo, Mammalian ; Gene Editing/methods/veterinary ; *Gene Knock-In Techniques/methods/veterinary ; Gene Targeting/methods/veterinary ; Sulfonamides/*pharmacology ; },
abstract = {Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single-stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.},
}

Animals
Animals, Genetically Modified
Benzamides/*pharmacology
CRISPR-Cas Systems/*drug effects/physiology
Cattle/*embryology
Cells, Cultured
DNA Breaks, Double-Stranded/drug effects
DNA End-Joining Repair/*drug effects
Embryo Culture Techniques
Embryo, Mammalian
Gene Editing/methods/veterinary
*Gene Knock-In Techniques/methods/veterinary
Gene Targeting/methods/veterinary
Sulfonamides/*pharmacology

@article {pmid31769791,
year = {2020},
author = {Sun, J and Wang, J and Zheng, D and Hu, X},
title = {Advances in therapeutic application of CRISPR-Cas9.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {164-174},
doi = {10.1093/bfgp/elz031},
pmid = {31769791},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Genetic Therapy ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing
Genetic Therapy
Humans

@article {pmid34281340,
year = {2021},
author = {Ma, L and Peng, L and Yin, L and Liu, G and Man, S},
title = {CRISPR-Cas12a-Powered Dual-Mode Biosensor for Ultrasensitive and Cross-validating Detection of Pathogenic Bacteria.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.1c00686},
pmid = {34281340},
issn = {2379-3694},
abstract = {Pathogenic bacteria infection severely threatens human health and causes substantial medical and financial concern. Rapid, sensitive, specific, and reliable detection of pathogenic bacteria is crucial. In the current study, a CRISPR-Cas12a-powered dual-mode biosensor was developed for ultrasensitive and cross-validating detection of pathogenic bacteria. Simply, the amplicons of Salmonella (used as a model)-specific invA sequence triggered CRISPR-Cas12a-based indiscriminate degradation of single-stranded DNAs that were supposed to link two gold nanoparticle (AuNP) probe pairs, inducing an aggregation-to-dispersion change. This generated observable color changes that became even more apparent after centrifugation. The color changes can be discerned by naked eyes and recorded using a portable colorimeter. Meanwhile, the photothermal effect of CRISPR-Cas12-powered AuNPs was explored for the first time through 808 nm near-infrared irradiation such that quantitative measurement can be carried out by recording temperatures using a thermal camera. For both modes, a limit of detection of 1 CFU/mL and a dynamic range of detection from 100 to 108 CFU/mL were obtained, which were comparable with or better than previously reported methods. Our proposed biosensor demonstrated satisfactory selectivity for Salmonella against other interfering cells. Furthermore, this biosensor proved to be capable of Salmonella detection in food samples. Regarding the real applications, the result from each mode can be used for cross-validation. Only the case having doubly confirmed positive or negative results can be assured, which rendered a more dependable biosensing conclusion. This technology not only expands the reach of the CRISPR-Cas system in biosensing but also provides a general method for bacteria sensing with desirable sensitivity, specificity, and cross-validating capacity.},
}

@article {pmid34257311,
year = {2021},
author = {Fareh, M and Zhao, W and Hu, W and Casan, JML and Kumar, A and Symons, J and Zerbato, JM and Fong, D and Voskoboinik, I and Ekert, PG and Rudraraju, R and Purcell, DFJ and Lewin, SR and Trapani, JA},
title = {Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4270},
pmid = {34257311},
issn = {2041-1723},
mesh = {Animals ; Antiviral Agents/pharmacology ; COVID-19/drug therapy/virology ; CRISPR-Cas Systems ; Chlorocebus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Development ; Genome, Viral ; HEK293 Cells ; Humans ; *Mutation ; SARS-CoV-2/genetics/*physiology ; Spike Glycoprotein, Coronavirus/genetics/metabolism ; Vero Cells ; Virus Replication/genetics/*physiology ; },
abstract = {The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.},
}

Animals
Antiviral Agents/pharmacology
COVID-19/drug therapy/virology
CRISPR-Cas Systems
Chlorocebus aethiops
Clustered Regularly Interspaced Short Palindromic Repeats
Drug Development
Genome, Viral
HEK293 Cells
Humans
*Mutation
SARS-CoV-2/genetics/*physiology
Spike Glycoprotein, Coronavirus/genetics/metabolism
Vero Cells
Virus Replication/genetics/*physiology

@article {pmid34244519,
year = {2021},
author = {Zhu, C and Wu, J and Sun, H and Briganti, F and Meder, B and Wei, W and Steinmetz, LM},
title = {Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4203},
pmid = {34244519},
issn = {2041-1723},
mesh = {*Alternative Splicing ; CRISPR-Cas Systems/genetics ; Cardiomyopathy, Dilated/*genetics/pathology ; Cell Differentiation/genetics ; Cell Line ; Feasibility Studies ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells ; Mitochondrial Proteins/genetics ; Molecular Sequence Annotation ; Muscle Proteins/genetics ; Mutation ; Myocytes, Cardiac/*pathology ; RNA Isoforms/genetics ; RNA, Guide/genetics ; RNA-Binding Proteins/*genetics ; RNA-Seq/*methods ; },
abstract = {Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.},
}

*Alternative Splicing
CRISPR-Cas Systems/genetics
Cardiomyopathy, Dilated/*genetics/pathology
Cell Differentiation/genetics
Cell Line
Feasibility Studies
Gene Editing
Humans
Induced Pluripotent Stem Cells
Mitochondrial Proteins/genetics
Molecular Sequence Annotation
Muscle Proteins/genetics
Mutation
Myocytes, Cardiac/*pathology
RNA Isoforms/genetics
RNA, Guide/genetics
RNA-Binding Proteins/*genetics
RNA-Seq/*methods

@article {pmid34244505,
year = {2021},
author = {Schmidt, MJ and Gupta, A and Bednarski, C and Gehrig-Giannini, S and Richter, F and Pitzler, C and Gamalinda, M and Galonska, C and Takeuchi, R and Wang, K and Reiss, C and Dehne, K and Lukason, MJ and Noma, A and Park-Windhol, C and Allocca, M and Kantardzhieva, A and Sane, S and Kosakowska, K and Cafferty, B and Tebbe, J and Spencer, SJ and Munzer, S and Cheng, CJ and Scaria, A and Scharenberg, AM and Cohnen, A and Coco, WM},
title = {Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4219},
pmid = {34244505},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/isolation & purification ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors/administration & dosage/genetics ; HEK293 Cells ; Humans ; Macaca fascicularis ; Male ; Mice ; Parvovirinae/genetics ; Protein Engineering ; RNA, Guide/*genetics ; Ribonucleases ; Staphylococcus/*enzymology/genetics ; Substrate Specificity ; Usher Syndromes/genetics/therapy ; },
abstract = {Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.},
}

Animals
CRISPR-Associated Protein 9/*genetics/isolation & purification
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Disease Models, Animal
Gene Editing/*methods
Genetic Therapy/methods
Genetic Vectors/administration & dosage/genetics
HEK293 Cells
Humans
Macaca fascicularis
Male
Mice
Parvovirinae/genetics
Protein Engineering
RNA, Guide/*genetics
Ribonucleases
Staphylococcus/*enzymology/genetics
Substrate Specificity
Usher Syndromes/genetics/therapy

@article {pmid34100058,
year = {2021},
author = {Yin, K and Ding, X and Li, Z and Sfeir, MM and Ballesteros, E and Liu, C},
title = {Autonomous lab-on-paper for multiplexed, CRISPR-based diagnostics of SARS-CoV-2.},
journal = {Lab on a chip},
volume = {21},
number = {14},
pages = {2730-2737},
pmid = {34100058},
issn = {1473-0189},
support = {R01 CA214072/CA/NCI NIH HHS/United States ; R01 EB023607/EB/NIBIB NIH HHS/United States ; R61 AI154642/AI/NIAID NIH HHS/United States ; },
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Amplification Techniques ; *Pandemics ; RNA, Viral/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.},
}

*COVID-19
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Nucleic Acid Amplification Techniques
*Pandemics
RNA, Viral/genetics
SARS-CoV-2
Sensitivity and Specificity

@article {pmid33972728,
year = {2021},
author = {Wang, L and Xue, W and Zhang, H and Gao, R and Qiu, H and Wei, J and Zhou, L and Lei, YN and Wu, X and Li, X and Liu, C and Wu, J and Chen, Q and Ma, H and Huang, X and Cai, C and Zhang, Y and Yang, B and Yin, H and Yang, L and Chen, J},
title = {Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations.},
journal = {Nature cell biology},
volume = {23},
number = {5},
pages = {552-563},
pmid = {33972728},
issn = {1476-4679},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics/metabolism ; *Gene Editing/methods ; HEK293 Cells ; Humans ; Mice ; Mutation/*genetics ; Proprotein Convertase 9/*genetics/metabolism ; },
abstract = {The fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30-40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.},
}

Animals
CRISPR-Cas Systems/genetics
Cytidine Deaminase/genetics/metabolism
*Gene Editing/methods
HEK293 Cells
Humans
Mice
Mutation/*genetics
Proprotein Convertase 9/*genetics/metabolism

@article {pmid33939828,
year = {2021},
author = {Hwang, GH and Jeong, YK and Habib, O and Hong, SA and Lim, K and Kim, JS and Bae, S},
title = {PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.},
journal = {Nucleic acids research},
volume = {49},
number = {W1},
pages = {W499-W504},
pmid = {33939828},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing ; Humans ; Internet ; Mutation ; RNA/chemistry ; Sequence Alignment ; *Software ; },
abstract = {Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.},
}

*CRISPR-Cas Systems
Gene Editing/*methods
High-Throughput Nucleotide Sequencing
Humans
Internet
Mutation
RNA/chemistry
Sequence Alignment
*Software

@article {pmid33846636,
year = {2021},
author = {Leibowitz, ML and Papathanasiou, S and Doerfler, PA and Blaine, LJ and Sun, L and Yao, Y and Zhang, CZ and Weiss, MJ and Pellman, D},
title = {Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.},
journal = {Nature genetics},
volume = {53},
number = {6},
pages = {895-905},
pmid = {33846636},
issn = {1546-1718},
support = {F32 DK118822/DK/NIDDK NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; R01 CA213404/CA/NCI NIH HHS/United States ; K22 CA216319/CA/NCI NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/genetics ; Antigens, CD34/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Division ; Chromosomes, Human/genetics ; *Chromothripsis ; DNA Cleavage ; *Gene Editing ; Genome, Human ; Humans ; Micronucleus, Germline/genetics ; Tumor Suppressor Protein p53/metabolism ; },
abstract = {Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole-genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus, micronuclei and chromosome bridges, which initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be considered and monitored.},
}

Anemia, Sickle Cell/genetics
Antigens, CD34/metabolism
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems/*genetics
Cell Division
Chromosomes, Human/genetics
*Chromothripsis
DNA Cleavage
*Gene Editing
Genome, Human
Humans
Micronucleus, Germline/genetics
Tumor Suppressor Protein p53/metabolism

@article {pmid33839407,
year = {2021},
author = {Watanabe, M and Takahashi, Y and Hiura, K and Nakano, K and Okamura, T and Sasaki, H and Sasaki, N},
title = {A single amino acid substitution in PRKDC is a determinant of sensitivity to Adriamycin-induced renal injury in mouse.},
journal = {Biochemical and biophysical research communications},
volume = {556},
number = {},
pages = {121-126},
doi = {10.1016/j.bbrc.2021.03.150},
pmid = {33839407},
issn = {1090-2104},
mesh = {Albuminuria/chemically induced/complications ; *Amino Acid Substitution ; Animals ; Base Sequence ; Biomarkers ; CRISPR-Cas Systems ; DNA-Activated Protein Kinase/*genetics/metabolism ; DNA-Binding Proteins/*genetics/metabolism ; *Disease Models, Animal ; Doxorubicin/*pharmacology ; Female ; Kidney Diseases/*chemically induced/complications/metabolism/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mutation ; Renal Insufficiency/chemically induced/complications/metabolism/pathology ; },
abstract = {Adriamycin (ADR)-induced nephropathy is frequently utilized in rodent models of podocytopathy. However, the application of this model in mice is limited to a few strains, such as BALB/c mice. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-PrkdcR2140C, that possess the R2140C mutation. After administration of ADR, B6-PrkdcR2140C mice exhibited massive proteinuria and glomerular and renal tubular injuries. In addition, there was no significant difference in the severity between B6-PrkdcR2140C and BALB/c. These findings demonstrated that B6-PrkdcR2140C show ADR-induced nephropathy susceptibility at a similar level to BALB/c, and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In future studies, ADR-induced nephropathy may become applicable to various kinds of genetically modified mice with a B6 background by mating with B6-PrkdcR2140C.},
}

Albuminuria/chemically induced/complications
*Amino Acid Substitution
Animals
Base Sequence
Biomarkers
CRISPR-Cas Systems
DNA-Activated Protein Kinase/*genetics/metabolism
DNA-Binding Proteins/*genetics/metabolism
*Disease Models, Animal
Doxorubicin/*pharmacology
Female
Kidney Diseases/*chemically induced/complications/metabolism/pathology
Mice
Mice, Inbred BALB C
Mice, Inbred C57BL
Mutation
Renal Insufficiency/chemically induced/complications/metabolism/pathology

@article {pmid33051367,
year = {2020},
author = {Goldman-Pinkovich, A and Kannan, S and Nitzan-Koren, R and Puri, M and Pawar, H and Bar-Avraham, Y and McDonald, J and Sur, A and Zhang, WW and Matlashewski, G and Madhubala, R and Michaeli, S and Myler, PJ and Zilberstein, D},
title = {Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.},
journal = {mBio},
volume = {11},
number = {5},
pages = {},
pmid = {33051367},
issn = {2150-7511},
mesh = {Animals ; Arginine/*metabolism ; CRISPR-Cas Systems ; Female ; *Host-Parasite Interactions ; Leishmania/*growth & development/*metabolism ; Leishmaniasis/metabolism/parasitology ; Lysosomes/parasitology ; Macrophages/*parasitology/physiology ; Membrane Transport Proteins/genetics ; Mice ; Mice, Inbred BALB C ; Phagosomes/parasitology/physiology ; },
abstract = {Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.},
}

Animals
Arginine/*metabolism
CRISPR-Cas Systems
Female
*Host-Parasite Interactions
Leishmania/*growth & development/*metabolism
Leishmaniasis/metabolism/parasitology
Lysosomes/parasitology
Macrophages/*parasitology/physiology
Membrane Transport Proteins/genetics
Mice
Mice, Inbred BALB C
Phagosomes/parasitology/physiology

@article {pmid32977162,
year = {2020},
author = {Cook, A and Bernstein, E},
title = {A strike against indolent neuroblastoma.},
journal = {EBioMedicine},
volume = {60},
number = {},
pages = {103000},
pmid = {32977162},
issn = {2352-3964},
mesh = {Biomarkers, Tumor ; CRISPR-Cas Systems ; Disease Management ; Disease Susceptibility ; Gene Targeting ; Humans ; Molecular Targeted Therapy ; Mutation ; Neuroblastoma/diagnosis/*etiology/therapy ; X-linked Nuclear Protein/antagonists & inhibitors ; },
}

Biomarkers, Tumor
CRISPR-Cas Systems
Disease Management
Disease Susceptibility
Gene Targeting
Humans
Molecular Targeted Therapy
Mutation
Neuroblastoma/diagnosis/*etiology/therapy
X-linked Nuclear Protein/antagonists & inhibitors

@article {pmid32878930,
year = {2020},
author = {Marton, T and Maufrais, C and d'Enfert, C and Legrand, M},
title = {Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in Candida albicans.},
journal = {mSphere},
volume = {5},
number = {5},
pages = {},
pmid = {32878930},
issn = {2379-5042},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Candida albicans/*genetics ; Gene Editing/*methods ; *Genome, Fungal ; Genomics ; *Homologous Recombination ; RNA, Guide/genetics ; *Transformation, Genetic ; },
abstract = {Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the organism's genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as C. albicans) and polyploid organisms. The effects of unleashing this nuclease within the genome of C. albicans are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a C. albicans strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in C. albicans, regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in C. albicans appears to limit transformation-associated genome changes.IMPORTANCE Genome editing is essential to nearly all research studies aimed at gaining insight into the molecular mechanisms underlying various biological processes, including those in the opportunistic pathogen Candida albicans The adaptation of the CRISPR-Cas9 system greatly facilitates genome engineering in many organisms. However, our understanding of the effects of CRISPR-Cas9 technology on the biology of C. albicans is limited. In this study, we sought to compare the extents of transformation-induced genomic changes within strains engineered using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation methods. CRISPR-Cas9-dependent transformation allows one to simultaneously target both homologs and, importantly, appears less mutagenic in C. albicans, since strains engineered using CRISPR-Cas9 display an overall decrease in concomitant genomic changes.},
}

CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Candida albicans/*genetics
Gene Editing/*methods
*Genome, Fungal
Genomics
*Homologous Recombination
RNA, Guide/genetics
*Transformation, Genetic

@article {pmid34280549,
year = {2021},
author = {Wang, Y and Lecourieux, F and Zhang, R and Dai, Z and Lecourieux, D and Li, S and Liang, Z},
title = {Data Comparison and Software Design for Easy Selection and Application of CRISPR-based Genome Editing Systems in Plants.},
journal = {Genomics, proteomics & bioinformatics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.gpb.2019.05.008},
pmid = {34280549},
issn = {2210-3244},
abstract = {CRISPR-based genome editing systems have been successfully and effectively used in many organisms. However, only a few studies have reported the comparison between CRISPR/Cas9 and CRISPR/Cpf1 systems in the whole-genome applications. Although many web-based toolkits are available, there is still a shortage of comprehensive, user-friendly, and plant-specific CRISPR databases and desktop software. In this study, we identified and analyzed the similarities and differences between CRISPR/Cas9 and CRISPR/Cpf1 systems by considering the abundance of proto-spacer adjacent motif (PAM) sites, effects of GC content, optimal proto-spacer length, potential universality within the plant kingdom, PAM-rich region (PARR) inhibiting ratio, and effects of G-quadruplex (G-Q) structures. Using this information, we built a comprehensive CRISPR database (including 138 plant genome data sources, www.grapeworld.cn/pc/index.html), which provides search tools for the identification of CRISPR editing sites in both CRISPR/Cas9 and CRISPR/Cpf1 systems. We also developed a desktop software on the basis of Perl/TK tool, which facilitates and improves the detection and analysis of CRISPR editing sites at the whole-genome level on Linux and/or Windows platform. Therefore, this study provides helpful data and software for easy selection and application of CRISPR-based genome editing systems in plants.},
}