@article {pmid34571006,
year = {2021},
author = {Zhang, Y and Nishiyama, T and Olson, EN and Bassel-Duby, R},
title = {CRISPR/Cas Correction of Muscular Dystrophies.},
journal = {Experimental cell research},
volume = {},
number = {},
pages = {112844},
doi = {10.1016/j.yexcr.2021.112844},
pmid = {34571006},
issn = {1090-2422},
abstract = {Muscular dystrophies are a heterogeneous group of monogenic neuromuscular disorders which lead to progressive muscle loss and degeneration of the musculoskeletal system. The genetic causes of muscular dystrophies are well characterized, but no effective treatments have been developed so far. The discovery and application of the CRISPR/Cas system for genome editing offers a new path for disease treatment with the potential to permanently correct genetic mutations. The post-mitotic and multinucleated features of skeletal muscle provide an ideal target for CRISPR/Cas therapeutic genome editing because correction of a subpopulation of nuclei can provide benefit to the whole myofiber. In this review, we provide an overview of the CRISPR/Cas system and its derivatives in genome editing, proposing potential CRISPR/Cas-based therapies to correct diverse muscular dystrophies, and we discuss challenges for translating CRISPR/Cas genome editing to a viable therapy for permanent correction of muscular dystrophies.},
}

@article {pmid34567840,
year = {2021},
author = {Wongpayak, P and Meesungnoen, O and Saejang, S and Subsoontorn, P},
title = {A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection.},
journal = {PeerJ},
volume = {9},
number = {},
pages = {e11996},
doi = {10.7717/peerj.11996},
pmid = {34567840},
issn = {2167-8359},
abstract = {The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.},
}

@article {pmid34563399,
year = {2021},
author = {Christie, KA and Kleinstiver, BP},
title = {Making the cut with PAMless CRISPR-Cas enzymes.},
journal = {Trends in genetics : TIG},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tig.2021.09.002},
pmid = {34563399},
issn = {0168-9525},
abstract = {Genome editing technologies simplify our ability to rewrite genetic blueprints of life. However, CRISPR-Cas enzymes found in nature can only manipulate a fraction of the genome. To overcome this limitation, new Cas variants have been developed that unlock nearly the entire genome for editing.},
}

@article {pmid34232061,
year = {2021},
author = {Chen, V and Griffin, ME and Maguin, P and Varble, A and Hang, HC},
title = {RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {18},
pages = {e0084421},
pmid = {34232061},
issn = {1098-5336},
support = {T32 AI070084/AI/NIAID NIH HHS/United States ; 2R01GM087544-10//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM087544/GM/NIGMS NIH HHS/United States ; 1R21AI156674-01//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (DIR, NIAID)/ ; 5R01CA245292-02//HHS | NIH | National Cancer Institute (NCI)/ ; R21 AI156674/AI/NIAID NIH HHS/United States ; R01 GM103593/GM/NIGMS NIH HHS/United States ; R01 CA245292/CA/NCI NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Enterococcus faecium/*genetics ; *Gene Editing ; Recombinases/*genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlying its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.},
}

Bacterial Proteins/*genetics
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems
Enterococcus faecium/*genetics
*Gene Editing
Recombinases/*genetics
Streptococcus pyogenes/genetics

@article {pmid34081824,
year = {2021},
author = {An, HW and Kim, SY and Kwon, JW and Seok, SH and Woo, SH and Kim, DY and Park, JW},
title = {In vivo CRISPR-Cas9 knockout screening using quantitative PCR identifies thymosin beta-4 X-linked that promotes diffuse-type gastric cancer metastasis.},
journal = {Molecular carcinogenesis},
volume = {60},
number = {9},
pages = {597-606},
doi = {10.1002/mc.23326},
pmid = {34081824},
issn = {1098-2744},
mesh = {Animals ; *Biomarkers, Tumor ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Expression ; *Gene Knockout Techniques ; Humans ; Mice ; Neoplasm Metastasis ; *Real-Time Polymerase Chain Reaction ; Signal Transduction ; Stomach Neoplasms/*genetics/*pathology ; Thymosin/*genetics ; },
abstract = {Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR-Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Pooled knockout mouse DGC cells were transplanted into a spleen of syngeneic immunocompetent mice to study clonal advantages in context of a complex process of liver metastasis. Tmsb4x (thymosin beta-4 X-linked), Hmox1, Ifitm3, Ldhb, and Itgb7 were identified as strong candidate genes that promote metastasis. In particular, Tmsb4x enhanced DGC metastasis and stomach organoid-generated tumor growth in in vivo transplantation models. Tmsb4x promoted tumor clonogenicity and anoikis resistance. In situ hybridization analysis showed that Tmsb4x is highly expressed in E-cadherin-negative mouse DGC models compared with mouse IGC and intestinal cancer models. E-cadherin deficiency also increased Tmsb4x expression in stomach organoids via Wnt signaling activation. Collectively, these results demonstrate that Tmsb4x promotes DGC metastasis. In addition, this experimental system will aid in the identification of novel target genes responsible for DGC metastasis.},
}

Animals
*Biomarkers, Tumor
*CRISPR-Cas Systems
Disease Models, Animal
Gene Expression
*Gene Knockout Techniques
Humans
Mice
Neoplasm Metastasis
*Real-Time Polymerase Chain Reaction
Signal Transduction
Stomach Neoplasms/*genetics/*pathology
Thymosin/*genetics

@article {pmid32159216,
year = {2020},
author = {Warma, A and Ndiaye, K},
title = {Functional effects of Tribbles homolog 2 in bovine ovarian granulosa cells†.},
journal = {Biology of reproduction},
volume = {102},
number = {6},
pages = {1177-1190},
doi = {10.1093/biolre/ioaa030},
pmid = {32159216},
issn = {1529-7268},
mesh = {Animals ; Antibodies ; CRISPR-Cas Systems ; *Cattle ; Connective Tissue Growth Factor/genetics/metabolism ; Female ; Granulosa Cells ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mitogen-Activated Protein Kinase 1/genetics/metabolism ; Mitogen-Activated Protein Kinase 3/genetics/metabolism ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; },
abstract = {Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6 h through 24 h post-hCG as compared to 0 h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.},
}

Animals
Antibodies
CRISPR-Cas Systems
*Cattle
Connective Tissue Growth Factor/genetics/metabolism
Female
Granulosa Cells
Intracellular Signaling Peptides and Proteins/genetics/*metabolism
Mitogen-Activated Protein Kinase 1/genetics/metabolism
Mitogen-Activated Protein Kinase 3/genetics/metabolism
Protein-Serine-Threonine Kinases/genetics/*metabolism

@article {pmid31653644,
year = {2020},
author = {De Rosa, L and Latella, MC and Secone Seconetti, A and Cattelani, C and Bauer, JW and Bondanza, S and De Luca, M},
title = {Toward Combined Cell and Gene Therapy for Genodermatoses.},
journal = {Cold Spring Harbor perspectives in biology},
volume = {12},
number = {5},
pages = {},
doi = {10.1101/cshperspect.a035667},
pmid = {31653644},
issn = {1943-0264},
mesh = {Animals ; CRISPR-Cas Systems ; Epidermis/metabolism ; Epidermolysis Bullosa/therapy ; Epidermolysis Bullosa Dystrophica/therapy ; Epidermolysis Bullosa, Junctional/therapy ; Genes, Dominant ; Genes, Recessive ; Genetic Therapy/*methods ; Genetic Vectors ; Humans ; Lentivirus/genetics ; Netherton Syndrome/therapy ; Retroviridae/genetics ; Simplexvirus ; Skin/metabolism ; Skin Diseases/*genetics/*therapy ; Stem Cells/cytology ; },
abstract = {To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.},
}

Animals
CRISPR-Cas Systems
Epidermis/metabolism
Epidermolysis Bullosa/therapy
Epidermolysis Bullosa Dystrophica/therapy
Epidermolysis Bullosa, Junctional/therapy
Genes, Dominant
Genes, Recessive
Genetic Therapy/*methods
Genetic Vectors
Humans
Lentivirus/genetics
Netherton Syndrome/therapy
Retroviridae/genetics
Simplexvirus
Skin/metabolism
Skin Diseases/*genetics/*therapy
Stem Cells/cytology

@article {pmid34560185,
year = {2021},
author = {Kwak, JS and Kim, MS and Kim, KH},
title = {Harnessing snakehead rhabdovirus (SHRV) for gene editing by installment of CRISPR/Cas9 in viral genome.},
journal = {Virus research},
volume = {},
number = {},
pages = {198578},
doi = {10.1016/j.virusres.2021.198578},
pmid = {34560185},
issn = {1872-7492},
abstract = {As there is no risk of viral genome integration into host chromosome, cytoplasmic RNA viruses can be a safer vehicle to deliver CRISPR/Cas system. Snakehead rhabdovirus (SHRV) is a piscine RNA virus belonging to the family Rhabdoviridae, and, in the present study, we evaluated the availability of SHRV as a tool for CRISPR/Cas9 delivery in mammalian cells. SHRV was grown well in baby hamster kidney (BHK-21) cells at 28 °C, and the replication ability was greatly reduced by temperature up-shift to 37 °C. We rescued a recombinant SHRV that harboring not only the interferon regulatory factor 9 (IRF9) gene-targeting single-guide RNA (sgRNA) but also Cas9 gene in the genome using the reverse genetic technology. The IRF9 gene of BHK-21 cells was knocked-out by the infection with the IRF9 gene-targeting rSHRV. Moreover, the rSHRVs were sharply disappeared in the cells by elevating temperature to 37 °C, suggesting the possible regulation of knockout efficiency before virus infection-caused cell damage. Although further optimization researches are needed to enhance the editing efficiency using the recombinant SHRV, to our knowledge, this is the first report on the possible applicability of piscine RNA virus for the gene editing in mammalian cells.},
}

@article {pmid34558981,
year = {2021},
author = {Li, Y and Mikkelsen, K and Lluch I Grané, O and Wang, Z and Tang, Y and Jiao, X and Ingmer, H and Høyland-Kroghsbo, NM and Li, Q},
title = {Functional Characterization of Type III-A CRISPR-Cas in a Clinical Human Methicillin-R Staphylococcus aureus Strain.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2021.0046},
pmid = {34558981},
issn = {2573-1602},
abstract = {CRISPR with its cas genes is an adaptive immune system that protects prokaryotes against foreign genetic elements. The type III-A CRISPR-Cas system is rarely found in Staphylococcus aureus, and little is known about its function in S. aureus. Here, we describe the genome characteristics of the clinical methicillin-resistant S. aureus (MRSA) strain TZ0912, carrying a type III-A CRISPR-Cas system. Phylogenetic analysis of 35 reported CRISPR-Cas-positive S. aureus strains revealed that the CRISPR-Cas system is prevalent in CC8 clones (10/35) and is located in the staphylococcal cassette chromosome mec (SCCmec) V, which confers methicillin resistance. Plasmid transformation and phage infection assays reveal that the type III-A CRISPR-Cas system protects TZ0912 against foreign DNA with sequence homology to the spacers located in the CRISPR array. We observed that the CRISPR-Cas immune system could effectively protect MRSA against phage attacks in both liquid culture and solid medium. In accordance with previous reports, using RNA-seq analysis and plasmid transformation assays, we find that the crRNAs close to the leading sequence of the CRISPR array are more highly expressed and are more effective at directing plasmid elimination compared to the distant spacers. This study established a model for evaluating the efficiency of naive CRISPR-Cas system in MRSA against phage, which could contribute to future research on the function of CRISPR-Cas in clinical MRSA isolates and improve phage therapy against MRSA infections.},
}

@article {pmid34558964,
year = {2021},
author = {Uranga, M and Vazquez-Vilar, M and Orzáez, D and Daròs, JA},
title = {CRISPR-Cas12a Genome Editing at the Whole-Plant Level Using Two Compatible RNA Virus Vectors.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2021.0049},
pmid = {34558964},
issn = {2573-1602},
abstract = {The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.},
}

@article {pmid34555248,
year = {2021},
author = {Mao, Y and Wang, M and Zhao, Y and Huang, B and Wu, Q and Zheng, Q and Botella, JR and Zhu, JK},
title = {Expanding the target range of base editing in plants without loss of efficiency by blocking RNA-silencing.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13712},
pmid = {34555248},
issn = {1467-7652},
abstract = {Bacterial-derived CRISPR/Cas systems are versatile platforms to engineer site-specific gene editing tools. Compared to the canonical Cas9-mediated DNA cleavage systems, which induce a high-proportion of frame-shift mutations, the recently developed base editing (BE) tools allow more precise and predictable base substitutions within a CRISPR/Cas9-defined editing window. Initially, such tools made use of engineered cytosine deaminases or evolved adenine deaminases to catalyze base deamination when fused to a Cas9 nickase (nCas9) (Rees and Liu, 2018).},
}

@article {pmid34553495,
year = {2021},
author = {Kordyś, M and Sen, R and Warkocki, Z},
title = {Applications of the versatile CRISPR-Cas13 RNA targeting system.},
journal = {Wiley interdisciplinary reviews. RNA},
volume = {},
number = {},
pages = {e1694},
doi = {10.1002/wrna.1694},
pmid = {34553495},
issn = {1757-7012},
support = {UMO-2017/26/D/NZ1/00887 (SONATA-13)//National Science Centre of Poland/ ; UMO-2019/33/B/NZ1/02260 (OPUS-17)//National Science Centre of Poland/ ; },
abstract = {CRISPR-Cas are adaptable natural prokaryotic defense systems that act against invading viruses and plasmids. Among the six currently known major CRISPR-Cas types, the type VI CRISPR-Cas13 is the only one known to exclusively bind and cleave foreign RNA. Within the last couple of years, this system has been adapted to serve numerous, and sometimes not obvious, applications, including some that might be developed as effective molecular therapies. Indeed, Cas13 has been adapted to kill antibiotic-resistant bacteria. In a cell-free environment, Cas13 has been used in the development of highly specific, sensitive, multiplexing-capable, and field-adaptable detection tools. Importantly, Cas13 can be reprogrammed and applied to eukaryotes to either combat pathogenic RNA viruses or in the regulation of gene expression, facilitating the knockdown of mRNA, circular RNA, and noncoding RNA. Furthermore, Cas13 has been harnessed for in vivo RNA modifications including programmable regulation of alternative splicing, A-to-I and C to U editing, and m6A modifications. Finally, approaches allowing for the detection and characterization of RNA-interacting proteins have also been demonstrated. Here, we provide a comprehensive overview of the applications utilizing CRISPR-Cas13 that illustrate its versatility. We also discuss the most important limitations of the CRISPR-Cas13-based technologies, and controversies regarding them. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.},
}

@article {pmid34553468,
year = {2021},
author = {Wei, Y and Yang, Z and Zong, C and Wang, B and Ge, X and Tan, X and Liu, X and Tao, Z and Wang, P and Ma, C and Wan, Y and Li, J},
title = {Trans Single-Stranded DNA Cleavage Via CRISPR/Cas14a1 Activated by Target RNA without Destruction.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {},
doi = {10.1002/anie.202110384},
pmid = {34553468},
issn = {1521-3773},
abstract = {As a CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis / trans cleave single-stranded DNA (ssDNA). Here, we discover an un-reported capacity of Cas14a1, RNA can trigger the trans ssDNA cleavage. This Cas14a1-based RNA-activated detection platform (A mplification, T ranscription, C as 14a1-based RNA -activated trans ssDNA cleavage, ATC as -RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNA-activation. Using ATCas-RNA via a fluorophore quencher-labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100% accuracy with 25 milk samples. This platform can serve as a new tool for high efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.},
}

@article {pmid34551428,
year = {2021},
author = {Cheng, F and Wang, R and Yu, H and Liu, C and Yang, J and Xiang, H and Li, M},
title = {Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkab821},
pmid = {34551428},
issn = {1362-4962},
support = {31771381//National Natural Science Foundation of China/ ; XDA24020101//Chinese Academy of Sciences/ ; 2020090//CAS/ ; },
abstract = {Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.},
}

@article {pmid34546709,
year = {2021},
author = {Mahas, A and Wang, Q and Marsic, T and Mahfouz, MM},
title = {A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.1c00181},
pmid = {34546709},
issn = {2161-5063},
abstract = {Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes' RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.},
}

@article {pmid34546072,
year = {2021},
author = {Flores Ramos, S and Brugger, SD and Escapa, IF and Skeete, CA and Cotton, SL and Eslami, SM and Gao, W and Bomar, L and Tran, TH and Jones, DS and Minot, S and Roberts, RJ and Johnston, CD and Lemon, KP},
title = {Genomic Stability and Genetic Defense Systems in Dolosigranulum pigrum, a Candidate Beneficial Bacterium from the Human Microbiome.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0042521},
doi = {10.1128/mSystems.00425-21},
pmid = {34546072},
issn = {2379-5077},
abstract = {Dolosigranulum pigrum is positively associated with indicators of health in multiple epidemiological studies of human nasal microbiota. Knowledge of the basic biology of D. pigrum is a prerequisite for evaluating its potential for future therapeutic use; however, such data are very limited. To gain insight into D. pigrum's chromosomal structure, pangenome, and genomic stability, we compared the genomes of 28 D. pigrum strains that were collected across 20 years. Phylogenomic analysis showed closely related strains circulating over this period and closure of 19 genomes revealed highly conserved chromosomal synteny. Gene clusters involved in the mobilome and in defense against mobile genetic elements (MGEs) were enriched in the accessory genome versus the core genome. A systematic analysis for MGEs identified the first candidate D. pigrum prophage and insertion sequence. A systematic analysis for genetic elements that limit the spread of MGEs, including restriction modification (RM), CRISPR-Cas, and deity-named defense systems, revealed strain-level diversity in host defense systems that localized to specific genomic sites, including one RM system hot spot. Analysis of CRISPR spacers pointed to a wealth of MGEs against which D. pigrum defends itself. These results reveal a role for horizontal gene transfer and mobile genetic elements in strain diversification while highlighting that in D. pigrum this occurs within the context of a highly stable chromosomal organization protected by a variety of defense mechanisms. IMPORTANCE Dolosigranulum pigrum is a candidate beneficial bacterium with potential for future therapeutic use. This is based on its positive associations with characteristics of health in multiple studies of human nasal microbiota across the span of human life. For example, high levels of D. pigrum nasal colonization in adults predicts the absence of Staphylococcus aureus nasal colonization. Also, D. pigrum nasal colonization in young children is associated with healthy control groups in studies of middle ear infections. Our analysis of 28 genomes revealed a remarkable stability of D. pigrum strains colonizing people in the United States across a 20-year span. We subsequently identified factors that can influence this stability, including genomic stability, phage predators, the role of MGEs in strain-level variation, and defenses against MGEs. Finally, these D. pigrum strains also lacked predicted virulence factors. Overall, these findings add additional support to the potential for D. pigrum as a therapeutic bacterium.},
}

@article {pmid34546037,
year = {2021},
author = {Dong, J and Chen, C and Liu, Y and Zhu, J and Li, M and Rao, VB and Tao, P},
title = {Engineering T4 Bacteriophage for In Vivo Display by Type V CRISPR-Cas Genome Editing.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.1c00251},
pmid = {34546037},
issn = {2161-5063},
abstract = {Bacteriophage T4 has enormous potential for biomedical applications due to its large size, capsid architecture, and high payload capability for protein and DNA delivery. However, it is not very easy to genetically engineer its genome heavily modified by cytosine hydroxymethylation and glucosylation. The glucosyl hydroxymethyl cytosine (ghmC) genome of phage is completely resistant to most restriction endonucleases and exhibits various degrees of resistance to CRISPR-Cas systems. Here, we found that the type V CRISPR-Cas12a system, which shows efficient cleavage of ghmC-modified genome when compared to the type II CRISPR-Cas9 system, can be synergistically employed to generate recombinant T4 phages. Focused on surface display, we analyzed the ability of phage T4 outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein) to tether, in vivo, foreign peptides and proteins to T4 capsid. Our data show that while these could be successfully expressed and displayed during the phage infection, shorter peptides are present at a much higher copy number than full-length proteins. However, the copy number of the latter could be elevated by driving the expression of the transgene using the strong T7 RNA polymerase expression system. This CRISPR-inspired approach has the potential to expand the application of phages to various basic and translational research projects.},
}

@article {pmid34544122,
year = {2021},
author = {Seher, TD and Nguyen, N and Ramos, D and Bapat, P and Nobile, CJ and Sindi, SS and Hernday, AD},
title = {AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {9},
pages = {},
doi = {10.1093/g3journal/jkab216},
pmid = {34544122},
issn = {2160-1836},
support = {/NH/NIH HHS/United States ; //NIH/ ; //National Institute of Allergy and Infectious Diseases/ ; //NIAID/ ; /GM/NIGMS NIH HHS/United States ; R15AI137975/GM/NIGMS NIH HHS/United States ; //Pew Biomedical Scholar Award/ ; //Pew Charitable Trusts/ ; },
abstract = {CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however, targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an "AddTag" sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene-editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing.},
}

@article {pmid34542686,
year = {2021},
author = {Gupta, R and Kazi, TA and Dey, D and Ghosh, A and Ravichandiran, V and Swarnakar, S and Roy, S and Biswas, SR and Ghosh, D},
title = {CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
pmid = {34542686},
issn = {1432-0614},
support = {BT/PR26301/GET/119/258/2017//department of biotechnology , ministry of science and technology/ ; BT/P/Budget/RD-74/2017//department of science and technology, government of west bengal/ ; },
abstract = {The emergence of SARS-CoV-2 has brought the world to a standstill, and till date, effective treatments and diagnostics against this idiosyncratic pathogen are lacking. As compared to the standard WHO/CDC qPCR detection method, which consumes several hours for detection, CRISPR-based SHERLOCK, DETECTR, and FELUDA have emerged as rapid diagnostic tools for the detection of the RNA genome of SARS-CoV-2 within an hour with 100% accuracy, specificity, and sensitivity. These attributes of CRISPR-based detection technologies have taken themselves one step ahead of available detection systems and are emerging as an inevitable tool for quick detection of the virus. Further, the discovery of Cas13s nucleases and their orthologs has opened a new corridor for exploitation of Cas13s as an antiviral therapy against SARS-CoV-2 and other viral diseases. One such approach is Prophylactic Antiviral CRISPR in huMAN cells (PACMAN), which needs a long haul to bring into therapy. The approval of SHERLOCK as the first CRISPR-based SARS-CoV-2 test kit by the FDA, for emergency diagnosis of COVID-19 patients, has given positive hope to scientists that sooner human trials of CRISPR-based therapy will be ratified. In this review, we have extensively reviewed the present CRISPR-based approaches, challenges, and future prospects in the light of diagnostics and therapeutics against SARS-CoV-2. KEY POINTS: • The discovery of Cas12 and Cas13 siblings allowed scientists to detect the viral genes. • Cas13d's identification aided scientists in precisely cleaving the SARS-CoV-2 ssRNA. • CRISPR-Cas system acts as "molecular detector and antiviral proctor."},
}

@article {pmid34541453,
year = {2021},
author = {Liu, W and Li, L and Jiang, J and Wu, M and Lin, P},
title = {Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics.},
journal = {Precision clinical medicine},
volume = {4},
number = {3},
pages = {179-191},
pmid = {34541453},
issn = {2516-1571},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) are efficient tools for targeting specific genes for laboratory research, agricultural engineering, biotechnology, and human disease treatment. Cas9, by far the most extensively used gene-editing nuclease, has shown great promise for the treatment of hereditary diseases, viral infection, cancers, and so on. Recent reports have revealed that some other types of CRISPR-Cas systems may also have surprising potential to join the fray as gene-editing tools for various applications. Despite the rapid progress in basic research and clinical tests, some underlying problems present continuous, significant challenges, such as editing efficiency, relative difficulty in delivery, off-target effects, immunogenicity, etc. This article summarizes the applications of CRISPR-Cas from bench to bedside and highlights the current obstacles that may limit the usage of CRISPR-Cas systems as gene-editing toolkits in precision medicine and offer some viewpoints that may help to tackle these challenges and facilitate technical development. CRISPR-Cas systems, as a powerful gene-editing approach, will offer great hopes in clinical treatments for many individuals with currently incurable diseases.},
}

@article {pmid34537403,
year = {2021},
author = {Ricroch, AE and Martin-Laffon, J and Rault, B and Pallares, VC and Kuntz, M},
title = {Next biotechnological plants for addressing global challenges: The contribution of transgenesis and new breeding techniques.},
journal = {New biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.nbt.2021.09.001},
pmid = {34537403},
issn = {1876-4347},
abstract = {The aim of this survey is to identify and characterize new products in plant biotechnology since 2015, especially in relation to the advent of New Breeding Techniques (NBTs) such as gene editing based on the CRISPR-Cas system. Transgenic (gene transfer or gene silencing) and gene edited traits which are approved or marketed in at least one country, or which have a non-regulated status in the USA, are collected, as well as related patents worldwide. In addition, to shed light on potential innovation for Africa, field trials on the continent are examined. The compiled data are classified in application categories, including agronomic improvements, industrial use and medical use, namely production of recombinant therapeutic molecules or vaccines (including against Covid-19). The data indicate that gene editing appears to be an effective complement to 'classical' transgenesis, the use of which is not declining, rather than a replacement, a trend also observed in the patenting landscape. Nevertheless, increased use of gene editing is apparent. Compared to transgenesis, gene editing has increased the proportion of some crop species and decreased others amongst approved, non-regulated or marketed products. A similar differential trend is observed for breeding traits. Gene editing has also favored the emergence of new private companies. China, and prevalently its public sector, overwhelmingly dominates the patenting landscape, but not the approved/marketed one, which is dominated by the USA. The data point in the direction that regulatory environments will favor or discourage innovation.},
}

@article {pmid34534948,
year = {2021},
author = {van Dongen, JE and Berendsen, JTW and Eijkel, JCT and Segerink, LI},
title = {A CRISPR/Cas12a-assisted in vitro diagnostic tool for identification and quantification of single CpG methylation sites.},
journal = {Biosensors & bioelectronics},
volume = {194},
number = {},
pages = {113624},
doi = {10.1016/j.bios.2021.113624},
pmid = {34534948},
issn = {1873-4235},
abstract = {The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a-MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.},
}

@article {pmid34533605,
year = {2021},
author = {Pulido-Quetglas, C and Johnson, R},
title = {Designing libraries for pooled CRISPR functional screens of long noncoding RNAs.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {},
number = {},
pages = {},
pmid = {34533605},
issn = {1432-1777},
support = {4534-08-2018//helmut horten stiftung, swiss cancer research foundation/ ; 18/FRL/6194//science foundation ireland through future research leaders award/ ; },
abstract = {Human and other genomes encode tens of thousands of long noncoding RNAs (lncRNAs), the vast majority of which remain uncharacterised. High-throughput functional screening methods, notably those based on pooled CRISPR-Cas perturbations, promise to unlock the biological significance and biomedical potential of lncRNAs. Such screens are based on libraries of single guide RNAs (sgRNAs) whose design is critical for success. Few off-the-shelf libraries are presently available, and lncRNAs tend to have cell-type-specific expression profiles, meaning that library design remains in the hands of researchers. Here we introduce the topic of pooled CRISPR screens for lncRNAs and guide readers through the three key steps of library design: accurate annotation of transcript structures, curation of optimal candidate sets, and design of sgRNAs. This review is a starting point and reference for researchers seeking to design custom CRISPR screening libraries for lncRNAs.},
}

@article {pmid34529907,
year = {2021},
author = {Zeng, XX and Zeng, J and Zhu, B},
title = {Future generation of combined multimodal approach to treat brain glioblastoma multiforme and potential impact on micturition control.},
journal = {Reviews in the neurosciences},
volume = {},
number = {},
pages = {},
doi = {10.1515/revneuro-2021-0068},
pmid = {34529907},
issn = {2191-0200},
abstract = {Glioblastoma remains lethal even when treated with standard therapy. This review aims to outline the recent development of various advanced therapeutics for glioblastoma and briefly discuss the potential impact of glioblastoma and some of its therapeutic approaches on the neurological function micturition control. Although immunotherapy led to success in treating hematological malignancies, but no similar success occurred in treatment for brain glioblastoma. Neither regenerative medicine nor stem cell therapy led to astounding success in glioblastoma. However, CRISPR Cas system holds potential in multiple applications due to its capacity to knock-in and knock-out genes, modify immune cells and cell receptors, which will enable it to address clinical challenges in immunotherapy such as CAR-T and regenerative therapy for brain glioblastoma, improving the precision and safety of these approaches. The studies mentioned in this review could indicate that glioblastoma is a malignant disease with multiple sophisticated barriers to be overcome and more challenges might arise in the attempt of researchers to yield a successful cure. A multimodal approach of future generation of refined and safe therapeutics derived from CRISPR Cas therapeutics, immunotherapy, and regenerative therapeutics mentioned in this review might prolong survival or even contribute towards a potential cure for glioblastoma.},
}

@article {pmid34529489,
year = {2021},
author = {Martin, TD and Patel, RS and Cook, DR and Choi, MY and Patil, A and Liang, AC and Li, MZ and Haigis, KM and Elledge, SJ},
title = {The adaptive immune system is a major driver of selection for tumor suppressor gene inactivation.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6561},
pages = {1327-1335},
doi = {10.1126/science.abg5784},
pmid = {34529489},
issn = {1095-9203},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Carcinogenesis ; Cell Line, Tumor ; Chemokine CCL2/metabolism ; Female ; GTP-Binding Protein alpha Subunits, G12-G13/genetics/metabolism ; *Gene Silencing ; *Genes, Tumor Suppressor ; Humans ; *Immune Evasion/genetics ; Mammary Neoplasms, Experimental/genetics/immunology/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, SCID ; Neoplasm Transplantation ; Neoplasms, Experimental/*genetics/*immunology/pathology ; Selection, Genetic ; Tumor Microenvironment ; },
abstract = {[Figure: see text].},
}

Animals
CRISPR-Cas Systems
*Carcinogenesis
Cell Line, Tumor
Chemokine CCL2/metabolism
Female
GTP-Binding Protein alpha Subunits, G12-G13/genetics/metabolism
*Gene Silencing
*Genes, Tumor Suppressor
Humans
*Immune Evasion/genetics
Mammary Neoplasms, Experimental/genetics/immunology/pathology
Mice
Mice, Inbred BALB C
Mice, Inbred C57BL
Mice, SCID
Neoplasm Transplantation
Neoplasms, Experimental/*genetics/*immunology/pathology
Selection, Genetic
Tumor Microenvironment

@article {pmid34529358,
year = {2021},
author = {Huang, RS and Lai, MC and Lin, S},
title = {Ex Vivo Expansion and CRISPR-Cas9 Genome Editing of Primary Human Natural Killer Cells.},
journal = {Current protocols},
volume = {1},
number = {9},
pages = {e246},
doi = {10.1002/cpz1.246},
pmid = {34529358},
issn = {2691-1299},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Knockout Techniques ; Humans ; Immunotherapy, Adoptive ; Killer Cells, Natural ; },
abstract = {Natural killer (NK) cells are potent innate immune cells that provide the surveillance and elimination of infected, stressed, and malignant cells. The unique immune recognition mechanisms and functions of NK cells make them an attractive cell type for immunology research and adoptive immunotherapy. However, primary NK cells are challenging to culture ex vivo and lack efficient genetic tools, hindering the research of NK cells and the development of NK cell therapeutics. Here we describe methods for the freeze-thaw process, feeder-free ex vivo expansion, CRISPR-Cas9 genome editing, and functional characterizations of primary human NK cells. Our protocol enables ∼30-fold and ∼2000-fold average expansion rates from 1 × 107 cryopreserved NK cells in 14 and 28 days, respectively. We also detail methods for CRISPR gene knockout and knockin by nucleofection of Cas9 ribonucleoproteins (RNP) and DNA repair templates. Gene knockout by Cas9 RNP nucleofection can be multiplexed to simultaneously target three genes. The CRISPR-edited cells can be cryopreserved and rethawed with high viability for future studies. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Thawing of natural killer cells Basic Protocol 2: Ex vivo expansion of natural killer cells Basic Protocol 3: Cryopreservation of expanded natural killer cells Basic Protocol 4: Characterization of natural killer cells: Flow cytometry and surface marker analysis Basic Protocol 5: Cytotoxicity and degranulation assays Basic Protocol 6: Preparation of homology-directed repair templates Basic Protocol 7: Nucleofection of CRISPR-Cas9 ribonucleoproteins Basic Protocol 8: Genotyping of gene-edited natural killer cells Basic Protocol 9: Phenotyping of gene-edited natural killer cells.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing
Gene Knockout Techniques
Humans
Immunotherapy, Adoptive
Killer Cells, Natural

@article {pmid34524446,
year = {2021},
author = {Hacker, L and Dorn, A and Enderle, J and Puchta, H},
title = {The repair of topoisomerase 2 cleavage complexes in Arabidopsis.},
journal = {The Plant cell},
volume = {},
number = {},
pages = {},
doi = {10.1093/plcell/koab228},
pmid = {34524446},
issn = {1532-298X},
abstract = {DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function non-redundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative non-homologous end joining (NHEJ) pathways. Double mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes non-ligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.},
}

@article {pmid34524200,
year = {2021},
author = {León, Y and Faherty, CS},
title = {Bacteriophages against enteropathogens: rediscovery and refinement of novel antimicrobial therapeutics.},
journal = {Current opinion in infectious diseases},
volume = {34},
number = {5},
pages = {491-499},
pmid = {34524200},
issn = {1473-6527},
support = {R21 AI146405/AI/NIAID NIH HHS/United States ; },
abstract = {PURPOSE OF REVIEW: Alarming rates of antibiotic resistance in bacteria and gastrointestinal dysbiosis associated with traditional antimicrobial therapy have led to renewed interests in developing bacteriophages as novel therapeutics. In this review, we highlight some of the recent advances in bacteriophage therapeutic development targeting important enteropathogens of the gastrointestinal tract.

RECENT FINDINGS: Bacteriophages are viruses that infect bacteria, either to utilize the bacterial machinery to produce new progeny or stably integrate into the bacterial chromosome to ensure maintenance of the viral genome. With recent advances in synthetic biology and the discovery of CRISPR-Cas systems used by bacteria to protect against bacteriophages, novel molecular applications are taking us beyond the discovery of bacteriophages and toward innovative applications, including the targeting of bacterial virulence factors, the use of temperate bacteriophages, and the production of bacteriophage proteins as antimicrobial agents. These technologies offer promise to target enteropathogens without disrupting the healthy microbiota of the gastrointestinal tract. Moreover, the use of nanoparticle technology and other modifications are helping researchers circumvent the harsh gastrointestinal conditions that could limit the efficacy of bacteriophages against enteric pathogens.

SUMMARY: This era of discovery and development offers significant potential to modify bacteriophages and overcome the global impact of enteropathogens.},
}

@article {pmid34522785,
year = {2021},
author = {Rao, GS and Jiang, W and Mahfouz, M},
title = {Synthetic directed evolution in plants: unlocking trait engineering and improvement.},
journal = {Synthetic biology (Oxford, England)},
volume = {6},
number = {1},
pages = {ysab025},
pmid = {34522785},
issn = {2397-7000},
abstract = {Genetic variation accelerates adaptation and resilience and enables the survival of species in their changing environment. Increasing the genetic diversity of crop species is essential to improve their yield and enhance food security. Synthetic directed evolution (SDE) employs localized sequence diversification (LSD) of gene sequence and selection pressure to evolve gene variants with better fitness, improved properties and desired phenotypes. Recently, CRISPR-Cas-dependent and -independent technologies have been applied for LSD to mediate synthetic evolution in diverse species, including plants. SDE holds excellent promise to discover, accelerate and expand the range of traits of the value in crop species. Here, we highlight the efficient SDE approaches for the LSD of plant genes, selection strategies and critical traits for targeted improvement. We discuss the potential of emerging technologies, including CRISPR-Cas base editing, retron editing, EvolvR and prime editing, to establish efficient SDE in plants. Moreover, we cover CRISPR-Cas-independent technologies, including T7 polymerase editor for continuous evolution. We highlight the key challenges and potential solutions of applying SDE technologies to improve the plant traits of the value.},
}

@article {pmid34521192,
year = {2021},
author = {Wang, C and Han, C and Du, X and Guo, W},
title = {Versatile CRISPR-Cas12a-Based Biosensing Platform Modulated with Programmable Entropy-Driven Dynamic DNA Networks.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.1c01597},
pmid = {34521192},
issn = {1520-6882},
abstract = {In addition to their roles as revolutionary genome engineering tools, CRISPR-Cas systems are also highly promising candidates in the construction of biosensing systems and diagnostic devices, which have attracted significant attention recently. However, the CRISPR-Cas system cannot be directly applied in the sensing of non-nucleic acid targets, and the needs of synthesizing and storing different vulnerable guide RNA for different targets also increase the application and storage costs of relevant biosensing systems, and therefore restrict their widespread applications. To tackle these barriers, in this work, a versatile CRISPR-Cas12a-based biosensing platform was developed through the introduction of an enzyme-free and robust DNA reaction network, the entropy-driven dynamic DNA network. By programming the sequences of the system, the entropy-driven catalysis-based dynamic DNA network can respond to different types of targets, such as nucleic acids or proteins, and then activate the CRISPR-Cas12a to generate amplified signals. As a proof of concept, both nucleic acid targets (a DNA target with random sequence, T, and an RNA target, microRNA-21 (miR-21)) and a non-nucleic acid target (a protein target, thrombin) were chosen as model analytes to address the feasibility of the designed sensing platform, with detection limits at the pM level for the nucleic acid analytes (7.4 pM for the DNA target T and 25.5 pM for miR-21) and 0.4 nM for thrombin. In addition, the detection of miR-21 or thrombin in human serum samples further demonstrated the applicability of the proposed biosensing platform in real sample analysis.},
}

@article {pmid34520771,
year = {2021},
author = {Mirzaei, S and Paskeh, MDA and Hashemi, F and Zabolian, A and Hashemi, M and Entezari, M and Tabari, T and Ashrafizadeh, M and Raee, P and Aghamiri, S and Aref, AR and Leong, HC and Kumar, AP and Samarghandian, S and Zarrabi, A and Hushmandi, K},
title = {Long non-coding RNAs as new players in bladder cancer: Lessons from pre-clinical and clinical studies.},
journal = {Life sciences},
volume = {},
number = {},
pages = {119948},
doi = {10.1016/j.lfs.2021.119948},
pmid = {34520771},
issn = {1879-0631},
abstract = {The clinical management of bladder cancer (BC) has become an increasing challenge due to high incidence rate of BC, malignant behavior of cancer cells and drug resistance. The non-coding RNAs are considered as key factors involved in BC progression. The long non-coding RNAs (lncRNAs) are RNA molecules and do not encode proteins. They have more than 200 nucleotides in length and affect gene expression at epigenetic, transcriptional and post-transcriptional phases. The lncRNAs demonstrate abnormal expression in BC cells and tissues. The present aims to identifying lncRNAs with tumor-suppressor and tumor-promoting roles, and evaluating their roles as regulatory of growth and migration. Apoptosis, glycolysis and EMT are tightly regulated by lncRNAs in BC. Response of BC cells to cisplatin, doxorubicin and gemcitabine chemotherapy is modulated by lncRNAs. LncRNAs regulate immune cell infiltration in tumor microenvironment and affect response of BC cells to immunotherapy. Besides, lncRNAs are able to regulate microRNAs, STAT3, Wnt, PTEN and PI3K/Akt pathways in affecting both proliferation and migration of BC cells. Noteworthy, anti-tumor compounds and genetic tools such as siRNA, shRNA and CRISPR/Cas systems can regulate lncRNA expression in BC. Finally, lncRNAs and exosomal lncRNAs can be considered as potential diagnostic and prognostic tools in BC.},
}

@article {pmid34519839,
year = {2021},
author = {Kruglov, O and Johnson, LDS and Minic, A and Jordan, K and Uger, RA and Wong, M and Sievers, EL and Shou, Y and Akilov, OE},
title = {The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {},
number = {},
pages = {},
pmid = {34519839},
issn = {1432-0851},
abstract = {CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.},
}

@article {pmid34518669,
year = {2021},
author = {Molla, KA and Sretenovic, S and Bansal, KC and Qi, Y},
title = {Precise plant genome editing using base editors and prime editors.},
journal = {Nature plants},
volume = {7},
number = {9},
pages = {1166-1187},
pmid = {34518669},
issn = {2055-0278},
abstract = {The development of CRISPR-Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications.},
}

@article {pmid34517958,
year = {2021},
author = {Pickerill, ES and Bernstein, DA},
title = {CRISPR mediated genome editing, a tool to dissect RNA modification processes.},
journal = {Methods in enzymology},
volume = {658},
number = {},
pages = {435-452},
doi = {10.1016/bs.mie.2021.06.013},
pmid = {34517958},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; RNA/genetics ; RNA, Guide/genetics ; },
abstract = {Though over 100 distinct RNA modifications have been identified, the roles for many of these modifications in vivo remain unknown. Genome editing is one tool investigators are using to better understand the roles these modifications play and the consequences of their absence. In this chapter, we describe how CRISPR mediated genome editing can be used to interrogate the process of RNA modification in C. albicans. Furthermore, we discuss how the protocols described can be altered to meet experimental demands. The underlying theory on which these protocols are based are applicable to a variety of model systems. The protocols described utilize the widely used S. pyogenes Cas9, but the field of genome editing is quickly evolving. We discuss the recent developments of more flexible CRISPR systems that can target a greater number of sites in the genome. These and other advancements make CRISPR mediated genome editing a practical methodology to investigate RNA modification.},
}

*CRISPR-Cas Systems
*Gene Editing
RNA/genetics
RNA, Guide/genetics

@article {pmid34517842,
year = {2021},
author = {Luo, Y and Na, R and Nowak, JS and Qiu, Y and Lu, QS and Yang, C and Marsolais, F and Tian, L},
title = {Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing.},
journal = {BMC plant biology},
volume = {21},
number = {1},
pages = {419},
pmid = {34517842},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/genetics/virology ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Gene Expression Regulation, Viral ; Genome, Plant ; Host-Pathogen Interactions/*genetics ; Mutagenesis ; Plant Viruses/*genetics ; RNA, Guide ; RNA, Plant ; RNA, Viral ; Soybeans/*genetics/*virology ; Virus Diseases/*genetics ; },
abstract = {BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing.

RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected.

CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.},
}

CRISPR-Cas Systems/*genetics
Crops, Agricultural/genetics/virology
Gene Editing/*methods
Gene Expression Regulation, Plant
Gene Expression Regulation, Viral
Genome, Plant
Host-Pathogen Interactions/*genetics
Mutagenesis
Plant Viruses/*genetics
RNA, Guide
RNA, Plant
RNA, Viral
Soybeans/*genetics/*virology
Virus Diseases/*genetics

@article {pmid34517762,
year = {2021},
author = {Wang, L and Sola, I and Enjuanes, L and Zuñiga, S},
title = {MOV10 Helicase Interacts with Coronavirus Nucleocapsid Protein and Has Antiviral Activity.},
journal = {mBio},
volume = {},
number = {},
pages = {e0131621},
doi = {10.1128/mBio.01316-21},
pmid = {34517762},
issn = {2150-7511},
abstract = {Coronaviruses (CoVs) are emergent pathogens that may cause life-threatening respiratory diseases in humans. Understanding of CoV-host interactions may help to identify novel therapeutic targets. MOV10 is an RNA helicase involved in different steps of cellular RNA metabolism. Both MOV10 antiviral and proviral activities have been described in a limited number of viruses, but this protein has not been previously associated with CoVs. We found that during Middle East respiratory syndrome coronavirus (MERS-CoV) infection, MOV10 aggregated in cytoplasmic structures colocalizing with viral nucleocapsid (N) protein. MOV10-N interaction was confirmed by endogenous MOV10 coimmunoprecipitation, and the presence of other cellular proteins was also detected in MOV10 complexes. MOV10 silencing significantly increased both N protein accumulation and virus titer, with no changes in the accumulation of viral RNAs. Moreover, MOV10 overexpression caused a 10-fold decrease in viral titers. These data indicated that MOV10 has antiviral activity during MERS-CoV infection. We postulated that this activity could be mediated by viral RNA sequestration, and in fact, RNA immunoprecipitation data showed the presence of viral RNAs in the MOV10 cytoplasmic complexes. Expression of wild-type MOV10 or of a MOV10 mutant without helicase activity in MOV10 knockout cell lines, developed by CRISPR-Cas technology, indicated that the helicase activity of MOV10 was required for its antiviral effect. Interestingly MOV10-N interaction was conserved in other mildly or highly pathogenic human CoVs, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although MOV10 antiviral activity was found only in highly pathogenic CoVs, suggesting a potential role of MOV10 in the modulation of human CoVs pathogenesis. IMPORTANCE Coronaviruses (CoVs) are emerging pathogens causing life-threatening diseases in humans. Knowledge of virus-host interactions and viral subversion mechanisms of host pathways is required for the development of effective countermeasures against CoVs. The interaction between cellular RNA helicase MOV10 and nucleocapsid (N) protein from several human CoVs is shown. Using MERS-CoV as a model, we demonstrate that MOV10 has antiviral function, requiring its helicase activity, most likely mediated by viral RNA sequestration in cytoplasmic ribonucleoprotein structures. Furthermore, we found that MOV10 antiviral activity may act only in highly pathogenic human CoVs, suggesting a role for MOV10 in modulating CoVs pathogenesis. The present study uncovers a complex network of viral and cellular RNAs and proteins interaction modulating the antiviral response against CoVs.},
}

@article {pmid34517092,
year = {2021},
author = {Savadi, S and Mangalassery, S and Sandesh, MS},
title = {Advances in genomics and genome editing for breeding next generation of fruit and nut crops.},
journal = {Genomics},
volume = {113},
number = {6},
pages = {3718-3734},
doi = {10.1016/j.ygeno.2021.09.001},
pmid = {34517092},
issn = {1089-8646},
abstract = {Fruit tree crops are an essential part of the food production systems and are key to achieve food and nutrition security. Genetic improvement of fruit trees by conventional breeding has been slow due to the long juvenile phase. Advancements in genomics and molecular biology have paved the way for devising novel genetic improvement tools like genome editing, which can accelerate the breeding of these perennial crops to a great extent. In this article, advancements in genomics of fruit trees covering genome sequencing, transcriptome sequencing, genome editing technologies (GET), CRISPR-Cas system based genome editing, potential applications of CRISPR-Cas9 in fruit tree crops improvement, the factors influencing the CRISPR-Cas editing efficiency and the challenges for CRISPR-Cas9 applications in fruit tree crops improvement are reviewed. Besides, base editing, a recently emerging more precise editing system, and the future perspectives of genome editing in the improvement of fruit and nut crops are covered.},
}

@article {pmid34516103,
year = {2021},
author = {Xia, X and Ma, B and Zhang, T and Lu, Y and Khan, MR and Hu, Y and Lei, C and Deng, S and He, Q and He, G and Zhang, K and Deng, R},
title = {G-Quadruplex-Probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization In Vivo.},
journal = {ACS sensors},
volume = {6},
number = {9},
pages = {3295-3302},
doi = {10.1021/acssensors.1c01061},
pmid = {34516103},
issn = {2379-3694},
abstract = {Foodborne pathogen infection is a key issue of food safety. Herein, we developed a label-free assay for Salmonella enterica (S. enterica) detection based on the G-quadruplex-probing CRISPR-Cas12 system (termed G-CRISPR-Cas), allowing highly sensitive detection of S. enterica and investigation of their colonization in chickens. The introduction of the G-quadruplex probe serving as the substrate of Cas 12a realized a label-free analysis for foodborne pathogens. Due to the amplification process induced by loop-mediated isothermal amplification (LAMP), G-CRISPR-Cas assay can detect S. enterica as low as 20 CFU. Specificity for pathogenic gene detection was guaranteed by the dual recognition process via LAMP primers and Cas 12a-guided RNA binding. The G-CRISPR-Cas assay was applied to explore S. enterica colonization in the intestinal tract and organs of chickens and showed the risk of S. enterica infection outside of the intestinal tract. The G-CRISPR-Cas assay is promising for on-site diagnosis of the infection or contamination of foodborne pathogens outside the laboratories, such as abattoirs and markets.},
}

@article {pmid34514290,
year = {2021},
author = {Jordan, WT and Currie, S and Schmitz, RJ},
title = {Multiplex genome editing in Arabidopsis thaliana using Mb3Cas12a.},
journal = {Plant direct},
volume = {5},
number = {9},
pages = {e344},
pmid = {34514290},
issn = {2475-4455},
abstract = {The use of CRISPR-Cas proteins for the creation of multiplex genome engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR-Cas9 system for multiplex genome engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome editing in Arabidopsis thaliana. We identified optimized cis-regulatory sequences for driving expression of single-transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex genome engineering in plant-based systems.},
}

@article {pmid34514084,
year = {2021},
author = {Silva, FSR and Erdogmus, E and Shokr, A and Kandula, H and Thirumalaraju, P and Kanakasabapathy, MK and Hardie, JM and Pacheco, LGC and Li, JZ and Kuritzkes, DR and Shafiee, H},
title = {SARS-CoV-2 RNA Detection by a Cellphone-Based Amplification-Free System with CRISPR/CAS-Dependent Enzymatic (CASCADE) Assay.},
journal = {Advanced materials technologies},
volume = {},
number = {},
pages = {2100602},
pmid = {34514084},
issn = {2365-709X},
support = {U54 HL119145/HL/NHLBI NIH HHS/United States ; },
abstract = {CRISPR (Clustered regularly interspaced short palindromic repeats)-based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS-CoV-2 molecular detection at the point of need. However, efficient translation of CRISPR-diagnostic technologies to field application is still hampered by dependence on target amplification and by reliance on fluorescence-based results readout. Herein, an amplification-free CRISPR/Cas12a-based diagnostic technology for SARS-CoV-2 RNA detection is presented using a smartphone camera for results readout. This method, termed Cellphone-based amplification-free system with CRISPR/CAS-dependent enzymatic (CASCADE) assay, relies on mobile phone imaging of a catalase-generated gas bubble signal within a microfluidic channel and does not require any external hardware optical attachments. Upon specific detection of a SARS-CoV-2 reverse-transcribed DNA/RNA heteroduplex target (orf1ab) by the ribonucleoprotein complex, the transcleavage collateral activity of the Cas12a protein on a Catalase:ssDNA probe triggers the bubble signal on the system. High analytical sensitivity in signal detection without previous target amplification (down to 50 copies µL-1) is observed in spiked samples, in ≈71 min from sample input to results readout. With the aid of a smartphone vision tool, high accuracy (AUC = 1.0; CI: 0.715 - 1.00) is achieved when the CASCADE system is tested with nasopharyngeal swab samples of PCR-positive COVID-19 patients.},
}

@article {pmid34511901,
year = {2021},
author = {Piperno, A and Sciortino, MT and Giusto, E and Montesi, M and Panseri, S and Scala, A},
title = {Recent Advances and Challenges in Gene Delivery Mediated by Polyester-Based Nanoparticles.},
journal = {International journal of nanomedicine},
volume = {16},
number = {},
pages = {5981-6002},
pmid = {34511901},
issn = {1178-2013},
abstract = {Gene therapy is a promising approach for the treatment of several diseases, such as chronic or viral infections, inherited disorders, and cancer. The cellular internalization of exogenous nucleic acids (NA) requires efficient delivery vehicles to overcome their inherent pharmacokinetic drawbacks, e.g. electrostatic repulsions, enzymatic degradation, limited cellular uptake, fast clearance, etc. Nanotechnological advancements have enabled the use of polymer-based nanostructured biomaterials as safe and effective gene delivery systems, in addition to viral vector delivery methods. Among the plethora of polymeric nanoparticles (NPs), this review will provide a comprehensive and in-depth summary of the polyester-based nanovehicles, including poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) NPs, used to deliver a variety of foreign NA, e.g. short interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The article will review the versatility of polyester-based nanocarriers including their recent application in the delivery of the clustered, regularly-interspaced, short palindromic repeats/Cas (CRISPR/Cas) genome editing system for treating gene-related diseases. The remaining challenges and future trend of the targeted delivery of this revolutionary genome-editing system will be discussed. Special attention will be given to the pivotal role of nanotechnology in tackling emerging infections such as coronavirus disease 2019 (COVID-19): ground-breaking mRNA vaccines delivered by NPs are currently used worldwide to fight the pandemic, pushing the boundaries of gene therapy.},
}

@article {pmid34509667,
year = {2021},
author = {Lee, BC and Lozano, RJ and Dunbar, CE},
title = {Understanding and overcoming adverse consequences of genome editing on hematopoietic stem and progenitor cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2021.09.001},
pmid = {34509667},
issn = {1525-0024},
abstract = {Hematopoietic stem and progenitor cell (HSPC) gene therapies have recently moved beyond gene-addition approaches to encompass targeted genome modification or correction, based on the development of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas technologies. Advances in ex vivo HSPC manipulation techniques have greatly improved HSPC susceptibility to genetic modification. Targeted gene-editing techniques enable precise modifications at desired genomic sites. Numerous preclinical studies have already demonstrated the therapeutic potential of gene therapies based on targeted editing. However, several significant hurdles related to adverse consequences of gene editing on HSPC function and genomic integrity remain before broad clinical potential can be realized. This review summarizes the status of HSPC gene editing, focusing on efficiency, genomic integrity, and long-term engraftment ability related to available genetic editing platforms and HSPC delivery methods. The response of long-term engrafting HSPCs to nuclease-mediated DNA breaks, with activation of p53, is a significant challenge, as are activation of innate and adaptive immune responses to editing components. Lastly, we propose alternative strategies that can overcome current hurdles to HSPC editing at various stages from cell collection to transplantation to facilitate successful clinical applications.},
}

@article {pmid34506127,
year = {2021},
author = {Feng, W and Peng, H and Xu, J and Liu, Y and Pabbaraju, K and Tipples, G and Joyce, MA and Saffran, HA and Tyrrell, DL and Babiuk, S and Zhang, H and Le, XC},
title = {Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.},
journal = {Analytical chemistry},
volume = {93},
number = {37},
pages = {12808-12816},
doi = {10.1021/acs.analchem.1c03456},
pmid = {34506127},
issn = {1520-6882},
mesh = {*COVID-19 ; COVID-19 Testing ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; Recombinases/genetics ; *Reverse Transcription ; SARS-CoV-2 ; Sensitivity and Specificity ; Technology ; },
abstract = {CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.},
}

*COVID-19
COVID-19 Testing
Humans
Nucleic Acid Amplification Techniques
RNA, Viral/genetics
Recombinases/genetics
*Reverse Transcription
SARS-CoV-2
Sensitivity and Specificity
Technology

@article {pmid34505742,
year = {2021},
author = {Verosloff, MS and Shapiro, SJ and Hawkins, EM and Alpay, E and Verma, D and Stanfield, EG and Kreindler, L and Jain, S and McKay, B and Hubbell, SA and Hendriks, CG and Blizard, BA and Broughton, JP and Chen, JS},
title = {CRISPR-Cas enzymes: The toolkit revolutionizing diagnostics.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2100304},
doi = {10.1002/biot.202100304},
pmid = {34505742},
issn = {1860-7314},
abstract = {The programmable nature of sequence-specific targeting by CRISPR-Cas nucleases has revolutionized a wide range of genomic applications and is now emerging as a method for nucleic acid detection. We explore how the diversity of CRISPR systems and their fundamental mechanisms have given rise to a wave of new methods for target recognition and readout. These cross-disciplinary advances found at the intersection of CRISPR biology and engineering have led to the ability to rapidly generate solutions for emerging global challenges like the COVID-19 pandemic. We further discuss the advances and potential for CRISPR-based detection to have an impact across a continuum of diagnostic applications. GRAPHICAL ABSTRACT AND LAY SUMMARY: The inherent programmability of CRISPR-Cas nucleases has enabled new methods for molecular diagnostics. Powered by the intersection of CRISPR biology and engineering, CRISPR-based diagnostics offer timely, accurate and actionable solutions across the continuum of testing applications.},
}

@article {pmid34505269,
year = {2021},
author = {Stellon, D and Tran, MTN and Talbot, J and Chear, S and Khalid, MKNM and Pébay, A and Vickers, JC and King, AE and Hewitt, AW and Cook, AL},
title = {CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {},
number = {},
pages = {},
pmid = {34505269},
issn = {1940-6029},
abstract = {Genetically encoded fluorescent biosensors (GEFBs) enable researchers to visualize and quantify cellular processes in live cells. Induced pluripotent stem cells (iPSCs) can be genetically engineered to express GEFBs via integration into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus. This can be achieved using CRISPR/Cas ribonucleoprotein targeting to cause a double-strand break at the AAVS1 locus, which subsequently undergoes homology-directed repair (HDR) in the presence of a donor plasmid containing the GEFB sequence. We describe an optimized protocol for CRISPR/Cas-mediated knock-in of GEFBs into the AAVS1 locus of human iPSCs that allows puromycin selection and which exhibits negligible off-target editing. The resulting iPSC lines can be differentiated into cells of different lineages while retaining expression of the GEFB, enabling live-cell interrogation of cell pathway activities across a diversity of disease models.},
}

@article {pmid34505086,
year = {2021},
author = {Ghanta, KS and Ishidate, T and Mello, CC},
title = {Microinjection for precision genome editing in Caenorhabditis elegans.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100748},
pmid = {34505086},
issn = {2666-1667},
support = {P40 OD010440/OD/NIH HHS/United States ; R37 GM058800/GM/NIGMS NIH HHS/United States ; },
abstract = {In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).},
}

@article {pmid34503839,
year = {2021},
author = {Toledo-Hernández, M and Lander, TA and Bao, C and Xie, K and Atta-Boateng, A and Wanger, TC},
title = {Genome-edited tree crops: mind the socioeconomic implementation gap.},
journal = {Trends in ecology & evolution},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tree.2021.08.007},
pmid = {34503839},
issn = {1872-8383},
abstract = {The discussion about CRISPR/Cas genome editing is focused mostly on technical aspects to improve productivity and climate resilience in major tree crops such as cocoa, coffee, and citrus. We suggest a solution to the largely ignored socioeconomic impacts for farmers, when new genome-edited varieties are introduced from the laboratory to the field.},
}

@article {pmid34502441,
year = {2021},
author = {Lyu, R and Ahmed, S and Fan, W and Yang, J and Wu, X and Zhou, W and Zhang, P and Yuan, L and Wang, H},
title = {Engineering Properties of Sweet Potato Starch for Industrial Applications by Biotechnological Techniques including Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502441},
issn = {1422-0067},
support = {2018YFD1000705, 2019YFD1000703，2019YFD1000701-2，2019YFD1000704-2//National Key R&D Program of China/ ; Y819V11111//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; G182402//Chenshan Special Fund for Shanghai Landscaping Administration Bureau Program/ ; 2019TQ0335//China Postdoctoral Science Foundation/ ; NA//Harold R. Burton Endowed Professorship/ ; NA//Postdoctoral fellowship from the University of Kentucky/ ; },
abstract = {Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes.},
}

@article {pmid34502143,
year = {2021},
author = {Arango, D and Bittar, A and Esmeral, NP and Ocasión, C and Muñoz-Camargo, C and Cruz, JC and Reyes, LH and Bloch, NI},
title = {Understanding the Potential of Genome Editing in Parkinson's Disease.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502143},
issn = {1422-0067},
support = {120484467666//Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS)/ ; },
abstract = {CRISPR is a simple and cost-efficient gene-editing technique that has become increasingly popular over the last decades. Various CRISPR/Cas-based applications have been developed to introduce changes in the genome and alter gene expression in diverse systems and tissues. These novel gene-editing techniques are particularly promising for investigating and treating neurodegenerative diseases, including Parkinson's disease, for which we currently lack efficient disease-modifying treatment options. Gene therapy could thus provide treatment alternatives, revolutionizing our ability to treat this disease. Here, we review our current knowledge on the genetic basis of Parkinson's disease to highlight the main biological pathways that become disrupted in Parkinson's disease and their potential as gene therapy targets. Next, we perform a comprehensive review of novel delivery vehicles available for gene-editing applications, critical for their successful application in both innovative research and potential therapies. Finally, we review the latest developments in CRISPR-based applications and gene therapies to understand and treat Parkinson's disease. We carefully examine their advantages and shortcomings for diverse gene-editing applications in the brain, highlighting promising avenues for future research.},
}

@article {pmid34498163,
year = {2021},
author = {Rao, MJ and Wang, L},
title = {CRISPR/Cas9 technology for improving agronomic traits and future prospective in agriculture.},
journal = {Planta},
volume = {254},
number = {4},
pages = {68},
pmid = {34498163},
issn = {1432-2048},
support = {2021M693797//china postdoctoral science foundation/ ; 31771775//National Outstanding Youth Foundation of China/ ; 31171524//National Outstanding Youth Foundation of China/ ; },
mesh = {Agriculture ; *CRISPR-Cas Systems/genetics ; Genome, Plant ; *Plant Breeding ; Plants, Genetically Modified/genetics ; Technology ; },
abstract = {MAIN CONCLUSION: In this review, we have focused on the CRISPR/Cas9 technology for improving the agronomic traits in plants through point mutations, knockout, and single base editing, and we highlighted the recent progress in plant metabolic engineering. CRISPR/Cas9 technology has immense power to reproduce plants with desired characters and revolutionizing the field of genome engineering by erasing the barriers in targeted genome editing. Agriculture fields are using this advance genome editing tool to get the desired traits in the crops plants such as increase yield, improve product quality attributes, and enhance resistance against biotic and abiotic stresses by identifying and editing genes of interest. This review focuses on CRISPR/Cas-based gene knockout for trait improvement and single base editing to boost yield, quality, stress tolerance, and disease resistance traits in crops. Use of CRISPR/Cas9 system to facilitate crop domestication and hybrid breeding are also touched. We summarize recent developments and up-gradation of delivery mechanism (nanotechnology and virus particle-based delivery system) and progress in multiplex gene editing. We also shed lights in advances and challenges of engineering the important metabolic pathways that contain a variety of dietary metabolites and phytochemicals. In addition, we endorsed substantial technical hurdles and possible ways to overcome the unpredictability of CRISPR/Cas technology for broader application across various crop species. We speculated that by making a strong interconnection among all genomic fields will give a gigantic bunt of knowledge to develop crop expressing desired traits.},
}

Agriculture
*CRISPR-Cas Systems/genetics
Genome, Plant
*Plant Breeding
Plants, Genetically Modified/genetics
Technology

@article {pmid34497366,
year = {2021},
author = {Zhou, Y and Zhang, L and Xie, YH and Wu, J},
title = {Advancements in detection of SARS-CoV-2 infection for confronting COVID-19 pandemics.},
journal = {Laboratory investigation; a journal of technical methods and pathology},
volume = {},
number = {},
pages = {},
pmid = {34497366},
issn = {1530-0307},
support = {81871997//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy.},
}

@article {pmid34496881,
year = {2021},
author = {Liang, J and Teng, P and Xiao, W and He, G and Song, Q and Zhang, Y and Peng, B and Li, G and Hu, L and Cao, D and Tang, Y},
title = {Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identification of SARS-CoV-2 from clinical samples.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {273},
pmid = {34496881},
issn = {1477-3155},
mesh = {COVID-19/diagnosis/virology ; CRISPR-Cas Systems/*genetics ; Gene Expression Regulation, Fungal ; Genes, Viral ; Humans ; *Nucleic Acid Amplification Techniques ; RNA, Viral/analysis ; SARS-CoV-2/*genetics/*isolation & purification ; Sensitivity and Specificity ; *Spectrum Analysis, Raman ; },
abstract = {The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).},
}

COVID-19/diagnosis/virology
CRISPR-Cas Systems/*genetics
Gene Expression Regulation, Fungal
Genes, Viral
Humans
*Nucleic Acid Amplification Techniques
RNA, Viral/analysis
SARS-CoV-2/*genetics/*isolation & purification
Sensitivity and Specificity
*Spectrum Analysis, Raman

@article {pmid34494480,
year = {2021},
author = {Amendola, M and Bedel, A and Buj Bello, A and Carrara, M and Concordet, JP and Frati, G and Gilot, D and Giovannangeli, C and Guttierrez, A and Laurent, M and Miccio, A and Moreau-Gaudry, F and Sourd, C and Valton, J and Verhoeyen, E},
title = {Recent progress in genome editing for gene therapy applications: the French perspective.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2021.191},
pmid = {34494480},
issn = {1557-7422},
abstract = {Recent advances in genome editing tools, especially the novel developments in the clustered regularly interspaced short palindrome repeats associated protein (CRISPR/Cas)-derived editing machinery have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome in order to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underlines their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing based therapy for a muscular dystrophy and the potency of T cell gene editing to increase anti-cancer activity of chimeric antigen receptor (CAR) T cells to combat cancer. We will also discuss technical obstacles and side-effects such as unwanted editing activity that need to be surmounted on the way towards a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.},
}

@article {pmid34493846,
year = {2021},
author = {},
title = {License CRISPR patents for free to share gene editing globally.},
journal = {Nature},
volume = {597},
number = {7875},
pages = {152},
doi = {10.1038/d41586-021-02420-x},
pmid = {34493846},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}

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

@article {pmid34490965,
year = {2021},
author = {Chen, HY and Hu, Y and Xu, XB and Zhou, YA and Li, NS and He, C and Xie, C and Lu, NH and Zhu, Y},
title = {Upregulation of oncogene Activin A receptor type I by Helicobacter pylori infection promotes gastric intestinal metaplasia via regulating CDX2.},
journal = {Helicobacter},
volume = {},
number = {},
pages = {e12849},
doi = {10.1111/hel.12849},
pmid = {34490965},
issn = {1523-5378},
support = {20194BCJ22016//Jiangxi Province/ ; 82000531//National Natural Science Foundation of China/ ; 8217033301//National Natural Science Foundation of China/ ; 20202BABL216006//the Youth Project of Jiangxi Natural Science Foundation/ ; GJJ190007//Key Fund of Jiangxi Education Department/ ; 202130191//Scientific Research of Health Commission of Jiangxi Province/ ; 2020A0047//Scientific Research of Traditional Chinese Medicine of Jiangxi Province/ ; PY201919//Young Teachers' Scientific Research and Cultivation Fund of the Medical Department of Nanchang University/ ; },
abstract = {BACKGROUND: Activin A receptor type I (ACVR1) is involved in tumorigenesis. However, the underlying molecular mechanisms of ACVR1 in gastric cancer (GC) and its association with Helicobacter pylori remained unclear.

MATERIALS AND METHODS: The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) database were utilized to explore the ACVR1 expression in GC and normal control and its association with survival. The ACVR1 was knocked out using CRISPR/Cas-9; RNA sequencing analysis was performed in AGS cells with ACVR1 knockout and normal control. Functional experiments (CCK-8, colony-forming, and transwell assays) were conducted to demonstrate the role of ACVR1 in cell proliferation, invasion, and metastasis. H. pylori-infected C57/BL6 models were established. ACVR1, p-Smad1/5, and CDX2 were detected in AGS cells cocultured with H. pylori strains. The CDX2 and key elements of BMP signaling pathway were detected in AGS cells with ACVR1 knockout and normal control. In addition, Immunohistochemistry was performed to detect the ACVR1 and CDX2 expression in gastric samples.

RESULTS: ACVR1 expression was higher in GC than normal control from TCGA, GEPIA, and samples collected from our hospital (p < 0.05). ACVR1 promoted cell proliferation, migration, and invasion in vitro. Both cagA+ and cagA- H. pylori could upregulate the expression ACVR1 (p < 0.05). Downregulation of ACVR1 inhibited the H. pylori-induced cell proliferation, migration, and invasion (p < 0.05). H. pylori increased the expression of p-Smad 1/5 and CDX2. The CDX2 and key elements of BMP signaling pathway were downregulated in AGS cells with ACVR1 knockout. ACVR1 and CDX2 were upregulated in the stage of intestinal metaplasia (IM). Moreover, ACVR1 and CDX2 expressions were higher in H. pylori-positive group than H. pylori-negative group (p < 0.05).

CONCLUSION: Our data indicate that H. pylori infection increases ACVR1 expression, promoting gastric IM via regulating CDX2, which is an essential step in H. pylori carcinogenesis.},
}

@article {pmid34489905,
year = {2021},
author = {Duan, C and Cao, H and Zhang, LH and Xu, Z},
title = {Harnessing the CRISPR-Cas Systems to Combat Antimicrobial Resistance.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {716064},
pmid = {34489905},
issn = {1664-302X},
abstract = {The emergence of antimicrobial-resistant (AMR) bacteria has become one of the most serious threats to global health, necessitating the development of novel antimicrobial strategies. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system, known as a bacterial adaptive immune system, can be repurposed to selectively target and destruct bacterial genomes other than invasive genetic elements. Thus, the CRISPR-Cas system offers an attractive option for the development of the next-generation antimicrobials to combat infectious diseases especially those caused by AMR pathogens. However, the application of CRISPR-Cas antimicrobials remains at a very preliminary stage and numerous obstacles await to be solved. In this mini-review, we summarize the development of using type I, type II, and type VI CRISPR-Cas antimicrobials to eradicate AMR pathogens and plasmids in the past a few years. We also discuss the most common challenges in applying CRISPR-Cas antimicrobials and potential solutions to overcome them.},
}

@article {pmid34489903,
year = {2021},
author = {Hoffert, M and Anderson, RE and Reveillaud, J and Murphy, LG and Stepanauskas, R and Huber, JA},
title = {Genomic Variation Influences Methanothermococcus Fitness in Marine Hydrothermal Systems.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {714920},
pmid = {34489903},
issn = {1664-302X},
abstract = {Hydrogenotrophic methanogens are ubiquitous chemoautotrophic archaea inhabiting globally distributed deep-sea hydrothermal vent ecosystems and associated subseafloor niches within the rocky subseafloor, yet little is known about how they adapt and diversify in these habitats. To determine genomic variation and selection pressure within methanogenic populations at vents, we examined five Methanothermococcus single cell amplified genomes (SAGs) in conjunction with 15 metagenomes and 10 metatranscriptomes from venting fluids at two geochemically distinct hydrothermal vent fields on the Mid-Cayman Rise in the Caribbean Sea. We observed that some Methanothermococcus lineages and their transcripts were more abundant than others in individual vent sites, indicating differential fitness among lineages. The relative abundances of lineages represented by SAGs in each of the samples matched phylogenetic relationships based on single-copy universal genes, and genes related to nitrogen fixation and the CRISPR/Cas immune system were among those differentiating the clades. Lineages possessing these genes were less abundant than those missing that genomic region. Overall, patterns in nucleotide variation indicated that the population dynamics of Methanothermococcus were not governed by clonal expansions or selective sweeps, at least in the habitats and sampling times included in this study. Together, our results show that although specific lineages of Methanothermococcus co-exist in these habitats, some outcompete others, and possession of accessory metabolic functions does not necessarily provide a fitness advantage in these habitats in all conditions. This work highlights the power of combining single-cell, metagenomic, and metatranscriptomic datasets to determine how evolution shapes microbial abundance and diversity in hydrothermal vent ecosystems.},
}

@article {pmid34489594,
year = {2021},
author = {Özcan, A and Krajeski, R and Ioannidi, E and Lee, B and Gardner, A and Makarova, KS and Koonin, EV and Abudayyeh, OO and Gootenberg, JS},
title = {Programmable RNA targeting with the single-protein CRISPR effector Cas7-11.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {34489594},
issn = {1476-4687},
abstract = {CRISPR-Cas interference is mediated by Cas effector nucleases that are either components of multisubunit complexes-in class 1 CRISPR-Cas systems-or domains of a single protein-in class 2 systems1-3. Here we show that the subtype III-E effector Cas7-11 is a single-protein effector in the class 1 CRISPR-Cas systems originating from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D. Cas7-11 from Desulfonema ishimotonii (DiCas7-11), when expressed in Escherichia coli, has substantial RNA interference effectivity against mRNAs and bacteriophages. Similar to many class 2 effectors-and unique among class 1 systems-DiCas7-11 processes pre-CRISPR RNA into mature CRISPR RNA (crRNA) and cleaves RNA at positions defined by the target:spacer duplex, without detectable non-specific activity. We engineered Cas7-11 for RNA knockdown and editing in mammalian cells. We show that Cas7-11 has no effects on cell viability, whereas other RNA-targeting tools (such as short hairpin RNAs and Cas13) show substantial cell toxicity4,5. This study illustrates the evolution of a single-protein effector from multisubunit class 1 effector complexes, expanding our understanding of the diversity of CRISPR systems. Cas7-11 provides the basis for new programmable RNA-targeting tools that are free of collateral activity and cell toxicity.},
}

@article {pmid34489579,
year = {2021},
author = {van der Oost, J and Fresco, LO},
title = {Waive CRISPR patents to meet food needs in low-income countries.},
journal = {Nature},
volume = {597},
number = {7875},
pages = {178},
doi = {10.1038/d41586-021-02397-7},
pmid = {34489579},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; Developing Countries/*economics ; *Food Supply/methods/standards ; Gene Editing/economics/*legislation & jurisprudence ; Humans ; Licensure/economics/*legislation & jurisprudence ; Patents as Topic/*legislation & jurisprudence ; Plant Breeding/economics/legislation & jurisprudence/methods ; Poverty ; },
}

*CRISPR-Cas Systems
Developing Countries/*economics
*Food Supply/methods/standards
Gene Editing/economics/*legislation & jurisprudence
Humans
Licensure/economics/*legislation & jurisprudence
Patents as Topic/*legislation & jurisprudence
Plant Breeding/economics/legislation & jurisprudence/methods
Poverty

@article {pmid34487299,
year = {2021},
author = {Guzmán, NM and Esquerra-Ruvira, B and Mojica, FJM},
title = {Digging into the lesser-known aspects of CRISPR biology.},
journal = {International microbiology : the official journal of the Spanish Society for Microbiology},
volume = {},
number = {},
pages = {},
pmid = {34487299},
issn = {1618-1905},
support = {PROMETEO/2017/129//conselleria d'educació, investigació, cultura i esport/ ; },
abstract = {A long time has passed since regularly interspaced DNA repeats were discovered in prokaryotes. Today, those enigmatic repetitive elements termed clustered regularly interspaced short palindromic repeats (CRISPR) are acknowledged as an emblematic part of multicomponent CRISPR-Cas (CRISPR associated) systems. These systems are involved in a variety of roles in bacteria and archaea, notably, that of conferring protection against transmissible genetic elements through an adaptive immune-like response. This review summarises the present knowledge on the diversity, molecular mechanisms and biology of CRISPR-Cas. We pay special attention to the most recent findings related to the determinants and consequences of CRISPR-Cas activity. Research on the basic features of these systems illustrates how instrumental the study of prokaryotes is for understanding biology in general, ultimately providing valuable tools for diverse fields and fuelling research beyond the mainstream.},
}

@article {pmid34486619,
year = {2021},
author = {Gong, H and Wu, Y and Zeng, R and Zeng, Y and Liu, X and Tang, D},
title = {CRISPR/Cas12a-mediated liposome-amplified strategy for the photoelectrochemical detection of nucleic acid.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {71},
pages = {8977-8980},
doi = {10.1039/d1cc03743a},
pmid = {34486619},
issn = {1364-548X},
abstract = {This study reports a photoelectrochemical biosensor for dopamine-loaded liposome-encoded magnetic beads cleaved by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas 12a system for the quantification of human papilloma virus (HPV)-related DNA using neodymium-doped BiOBr nanosheets (Nd-BiOBr) as a photoactive matrix. Magnetic beads and dopamine-loaded liposomes are covalently attached to the both ends of ssDNA to construct dumbbell-shaped dopamine-loaded liposome-encoded magnetic bead (DLL-MB) probes. When the guide RNA binds to the target HPV-16, the ssDNA will be cleaved by Cas12a, thereby degrading the double dumbbell probes. After magnetic separation, the dissolved DLLs are treated with Triton X-100 to release the dopamine (as an electron donor), which was then detected by an amplified photocurrent using the Nd-BiOBr-based photoelectrode.},
}

@article {pmid34486151,
year = {2021},
author = {Baliga, P and Shekar, M and Tg, P and Sk, G},
title = {Investigation into the prevalent CRISPR-Cas systems among the Aeromonas genus.},
journal = {Journal of basic microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1002/jobm.202100234},
pmid = {34486151},
issn = {1521-4028},
support = {PDF/2018/ 000811//Science and Engineering Research Board, India/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune system that checks invasion by mobile genetic elements through nuclease targeting. In this study, we investigated the occurrence, diversity, and features of the CRISPR system in the genus Aeromonas using bioinformatics tools. Only 13 out of 122 complete genomes (10.66%) of the genus Aeromonas from the NCBI GenBank database harbored the CRISPR system. The Type I-F system was the most prevalent CRISPR system among the Aeromonads, followed by the Type I-E system. Only one strain harbored a Type I-C CRISPR system. Among the Aeromonads, Aeromonas caviae (22.7%) and Aeromonas veronii (20%) had a higher prevalence rate of the complete CRISPR system. The analysis of direct repeat (DR) sequences showed that all could form stable RNA secondary structures. A phylogenetic tree generated for the Cas1 protein classified CRISPR subtypes into three distinct clusters. Among the 748 spacers investigated, 41.98% and 17.25% showed perfect homology to phage and plasmid sequences, respectively. Some arrays had duplicated spacers. The CRISPR loci are closely linked to antibiotic resistance genes in most strains. Collectively, our results would contribute to research on antibiotic resistance in the Aeromonas group, and provide new insights into the diversity and evolution of the CRISPR-Cas system.},
}

@article {pmid34485609,
year = {2021},
author = {Głów, D and Meyer, S and García Roldán, I and Akingunsade, LM and Riecken, K and Fehse, B},
title = {LATE-a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects.},
journal = {Molecular therapy. Methods & clinical development},
volume = {22},
number = {},
pages = {249-262},
pmid = {34485609},
issn = {2329-0501},
abstract = {Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.},
}

@article {pmid34485599,
year = {2021},
author = {Zhang, Y and Nishiyama, T and Li, H and Huang, J and Atmanli, A and Sanchez-Ortiz, E and Wang, Z and Mireault, AA and Mammen, PPA and Bassel-Duby, R and Olson, EN},
title = {A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation.},
journal = {Molecular therapy. Methods & clinical development},
volume = {22},
number = {},
pages = {122-132},
pmid = {34485599},
issn = {2329-0501},
abstract = {Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD.},
}

@article {pmid34485380,
year = {2021},
author = {Chen, CC and Guan, G and Qi, X and Abulaiti, A and Zhang, T and Liu, J and Lu, F and Chen, X},
title = {Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System.},
journal = {Frontiers in molecular biosciences},
volume = {8},
number = {},
pages = {676957},
pmid = {34485380},
issn = {2296-889X},
abstract = {The integration of HBV DNA is one of the carcinogenic mechanisms of HBV. The clearance of HBV integration in hepatocyte is of great significance to cure chronic HBV infection and thereby prevent the occurrence of HBV-related hepatocellular carcinoma (HCC). However, the low throughput of traditional methods, such as Alu-PCR, results in low detecting sensitivity of HBV integration. Although the second-generation sequencing can obtain a large amount of sequencing data, but the sequencing fragments are extremely short, so it cannot fully explore the characteristics of HBV integration. In this study, we used the third-generation sequencing technology owning advantages both in sequencing length and in sequencing depth to analyze the HBV integration characteristics in PLC/PRF/5 cells comprehensively. A total of 4,142,311 cleaning reads was obtained, with an average length of 18,775.6 bp, of which 84 reads were fusion fragments of the HBV DNA and human genome. These 84 fragments located in seven chromosomes, including chr3, chr4, chr8, chr12, chr13, chr16, and chr17. We observed lots of DNA rearrangement both in the human genome and in HBV DNA fragments surrounding the HBV integration site, indicating the genome instability causing by HBV integration. By analyzing HBV integrated fragments of PLC/PRF/5 cells that can potentially express HBsAg, we selected three combinations of sgRNAs targeting the integrated fragments to knock them out with CRISPR/Cas9 system. We found that the sgRNA combinations could significantly decrease the level of HBsAg in the supernatant of PLC/PRF/5 cells, while accelerated cell proliferation. This study proved the effectiveness of third-generation sequencing to detect HBV integration, and provide a potential strategy to reach HBsAg clearance for chronic HBV infection patients, but the knock-out of HBV integration from human genome by CRISPR/Cas9 system may have a potential of carcinogenic risk.},
}

@article {pmid34484175,
year = {2021},
author = {Dawes, JC and Uren, AG},
title = {Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {670280},
pmid = {34484175},
issn = {1664-3224},
abstract = {Cancer genome sequencing has identified dozens of mutations with a putative role in lymphomagenesis and leukemogenesis. Validation of driver mutations responsible for B cell neoplasms is complicated by the volume of mutations worthy of investigation and by the complex ways that multiple mutations arising from different stages of B cell development can cooperate. Forward and reverse genetic strategies in mice can provide complementary validation of human driver genes and in some cases comparative genomics of these models with human tumors has directed the identification of new drivers in human malignancies. We review a collection of forward genetic screens performed using insertional mutagenesis, chemical mutagenesis and exome sequencing and discuss how the high coverage of subclonal mutations in insertional mutagenesis screens can identify cooperating mutations at rates not possible using human tumor genomes. We also compare a set of independently conducted screens from Pax5 mutant mice that converge upon a common set of mutations observed in human acute lymphoblastic leukemia (ALL). We also discuss reverse genetic models and screens that use CRISPR-Cas, ORFs and shRNAs to provide high throughput in vivo proof of oncogenic function, with an emphasis on models using adoptive transfer of ex vivo cultured cells. Finally, we summarize mouse models that offer temporal regulation of candidate genes in an in vivo setting to demonstrate the potential of their encoded proteins as therapeutic targets.},
}

@article {pmid34482543,
year = {2021},
author = {Xu, H and Wali, R and Cheruiyot, C and Bodenschatz, J and Hasenfuss, G and Janshoff, A and Habeck, M and Ebert, A},
title = {Non-negative blind deconvolution for signal processing in a CRISPR-edited iPSC-cardiomyocyte model of dilated cardiomyopathy.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.14189},
pmid = {34482543},
issn = {1873-3468},
support = {F/13/20//Deutsche Stiftung fuer Herzforschung/ ; SFB1002//Deutsche Forschungsgemeinschaft/ ; },
abstract = {We developed an integrated platform for analysis of parameterized data from human disease models. We report a non-negative blind deconvolution (NNBD) approach to quantify calcium (Ca2+) handling, beating force and contractility in human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at the single-cell level. We employed CRISPR/Cas gene editing to introduce a dilated cardiomyopathy (DCM)-causing mutation in troponin T (TnT), TnT-R141W, into wild-type control iPSCs (MUT). The NNDB-based method enabled data parametrization, fitting and analysis in wild-type controls versus isogenic MUT iPSC-CMs. Of note, Cas9-edited TnT-R141W iPSC-CMs revealed significantly reduced beating force and prolonged contractile event duration. The NNBD-based platform provides an alternative framework for improved quantitation of molecular disease phenotypes and may contribute to the development of novel diagnostic tools.},
}

@article {pmid34480847,
year = {2021},
author = {Xu, X and Chemparathy, A and Zeng, L and Kempton, HR and Shang, S and Nakamura, M and Qi, LS},
title = {Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2021.08.008},
pmid = {34480847},
issn = {1097-4164},
abstract = {Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.},
}

@article {pmid34480322,
year = {2021},
author = {Orf, GS and Gisriel, CJ and Granstrom, J and Baker, PL and Redding, KE},
title = {The PshX subunit of the photochemical reaction center from Heliobacterium modesticaldum acts as a low-energy antenna.},
journal = {Photosynthesis research},
volume = {},
number = {},
pages = {},
pmid = {34480322},
issn = {1573-5079},
support = {DE-SC0010575//Basic Energy Sciences/ ; },
abstract = {The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. To assess the function of PshX, we produced a ∆pshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ∆pshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer toward the P800 special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.},
}

@article {pmid34479645,
year = {2021},
author = {Zhou, K and Xu, Y and Zhang, R and Qian, PY},
title = {Arms race in a cell: genomic, transcriptomic, and proteomic insights into intracellular phage-bacteria interplay in deep-sea snail holobionts.},
journal = {Microbiome},
volume = {9},
number = {1},
pages = {182},
pmid = {34479645},
issn = {2049-2618},
mesh = {Animals ; Bacteria/genetics ; *Bacteriophages/genetics ; Genomics ; Proteomics ; Snails ; Transcriptome/genetics ; },
abstract = {BACKGROUND: Deep-sea animals in hydrothermal vents often form endosymbioses with chemosynthetic bacteria. Endosymbionts serve essential biochemical and ecological functions, but the prokaryotic viruses (phages) that determine their fate are unknown.

RESULTS: We conducted metagenomic analysis of a deep-sea vent snail. We assembled four genome bins for Caudovirales phages that had developed dual endosymbiosis with sulphur-oxidising bacteria (SOB) and methane-oxidising bacteria (MOB). Clustered regularly interspaced short palindromic repeat (CRISPR) spacer mapping, genome comparison, and transcriptomic profiling revealed that phages Bin1, Bin2, and Bin4 infected SOB and MOB. The observation of prophages in the snail endosymbionts and expression of the phage integrase gene suggested the presence of lysogenic infection, and the expression of phage structural protein and lysozyme genes indicated active lytic infection. Furthermore, SOB and MOB appear to employ adaptive CRISPR-Cas systems to target phage DNA. Additional expressed defence systems, such as innate restriction-modification systems and dormancy-inducing toxin-antitoxin systems, may co-function and form multiple lines for anti-viral defence. To counter host defence, phages Bin1, Bin2, and Bin3 appear to have evolved anti-restriction mechanisms and expressed methyltransferase genes that potentially counterbalance host restriction activity. In addition, the high-level expression of the auxiliary metabolic genes narGH, which encode nitrate reductase subunits, may promote ATP production, thereby benefiting phage DNA packaging for replication.

CONCLUSIONS: This study provides new insights into phage-bacteria interplay in intracellular environments of a deep-sea vent snail. Video Abstract.},
}

Animals
Bacteria/genetics
*Bacteriophages/genetics
Genomics
Proteomics
Snails
Transcriptome/genetics

@article {pmid34478654,
year = {2021},
author = {Donohoue, PD and Pacesa, M and Lau, E and Vidal, B and Irby, MJ and Nyer, DB and Rotstein, T and Banh, L and Toh, MS and Gibson, J and Kohrs, B and Baek, K and Owen, ALG and Slorach, EM and van Overbeek, M and Fuller, CK and May, AP and Jinek, M and Cameron, P},
title = {Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.},
journal = {Molecular cell},
volume = {81},
number = {17},
pages = {3637-3649.e5},
doi = {10.1016/j.molcel.2021.07.035},
pmid = {34478654},
issn = {1097-4164},
abstract = {The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.},
}

@article {pmid34478558,
year = {2021},
author = {Lee, KZ and Mechikoff, MA and Kikla, A and Liu, A and Pandolfi, P and Fitzgerald, K and Gimble, FS and Solomon, KV},
title = {NgAgo possesses guided DNA nicking activity.},
journal = {Nucleic acids research},
volume = {49},
number = {17},
pages = {9926-9937},
doi = {10.1093/nar/gkab757},
pmid = {34478558},
issn = {1362-4962},
support = {41000622//Ralph W. and Grace M. Showalter Research Trust Fund/ ; S1041//National Institute of Food and Agriculture/ ; 60000025//Purdue Research Foundation/ ; },
abstract = {Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.},
}

@article {pmid34475857,
year = {2021},
author = {Liu, Q and Wang, S and Long, J and Chen, Z and Yang, B and Lin, F},
title = {Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {686715},
pmid = {34475857},
issn = {1664-302X},
abstract = {The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5'-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99A strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.},
}

@article {pmid34475598,
year = {2021},
author = {},
title = {Kazuto Kato: the ethics of editing humanity.},
journal = {Bulletin of the World Health Organization},
volume = {99},
number = {9},
pages = {616-617},
doi = {10.2471/BLT.21.030921},
pmid = {34475598},
issn = {1564-0604},
mesh = {*Bioethical Issues/history ; Biotechnology/*ethics ; CRISPR-Cas Systems ; Ethical Theory ; *Ethics, Medical/history ; Gene Editing/*ethics/history ; History, 21st Century ; Human Characteristics ; Humans ; },
abstract = {Gary Humphreys talks to Kazuto Kato about the ethical and societal challenges posed by biotechnologies that allow for the editing of the human genome.},
}

*Bioethical Issues/history
Biotechnology/*ethics
CRISPR-Cas Systems
Ethical Theory
*Ethics, Medical/history
Gene Editing/*ethics/history
History, 21st Century
Human Characteristics
Humans

@article {pmid34475565,
year = {2021},
author = {Wu, Z and Zhang, Y and Yu, H and Pan, D and Wang, Y and Wang, Y and Li, F and Liu, C and Nan, H and Chen, W and Ji, Q},
title = {Programmed genome editing by a miniature CRISPR-Cas12f nuclease.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {34475565},
issn = {1552-4469},
support = {2019M651627//China Postdoctoral Science Foundation/ ; 91753127//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2207783//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.},
}

@article {pmid34475519,
year = {2021},
author = {Bartlau, N and Wichels, A and Krohne, G and Adriaenssens, EM and Heins, A and Fuchs, BM and Amann, R and Moraru, C},
title = {Highly diverse flavobacterial phages isolated from North Sea spring blooms.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
pmid = {34475519},
issn = {1751-7370},
support = {INST 184/157-1 FUGG//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; FOR2406//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; FOR2406//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; FOR2406//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; FOR2406//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {It is generally recognized that phages are a mortality factor for their bacterial hosts. This could be particularly true in spring phytoplankton blooms, which are known to be closely followed by a highly specialized bacterial community. We hypothesized that phages modulate these dense heterotrophic bacteria successions following phytoplankton blooms. In this study, we focused on Flavobacteriia, because they are main responders during these blooms and have an important role in the degradation of polysaccharides. A cultivation-based approach was used, obtaining 44 lytic flavobacterial phages (flavophages), representing twelve new species from two viral realms. Taxonomic analysis allowed us to delineate ten new phage genera and ten new families, from which nine and four, respectively, had no previously cultivated representatives. Genomic analysis predicted various life styles and genomic replication strategies. A likely eukaryote-associated host habitat was reflected in the gene content of some of the flavophages. Detection in cellular metagenomes and by direct-plating showed that part of these phages were actively replicating in the environment during the 2018 spring bloom. Furthermore, CRISPR/Cas spacers and re-isolation during two consecutive years suggested that, at least part of the new flavophages are stable components of the microbial community in the North Sea. Together, our results indicate that these diverse flavophages have the potential to modulate their respective host populations.},
}

@article {pmid34473995,
year = {2021},
author = {Enríquez, P and Krajewski, K and Strahl, BD and Rothbart, SB and Dowen, RH and Rose, RB},
title = {Binding specificity and function of the SWI/SNF subunit SMARCA4 bromodomain interaction with acetylated histone H3K14.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {101145},
doi = {10.1016/j.jbc.2021.101145},
pmid = {34473995},
issn = {1083-351X},
support = {R35 GM124736/GM/NIGMS NIH HHS/United States ; R35 GM126900/GM/NIGMS NIH HHS/United States ; },
abstract = {Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD-H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small molecule BD inhibitors to treat cancers and other diseases.},
}

@article {pmid34472610,
year = {2021},
author = {Fujikawa, T and Ogura, Y and Ishigami, K and Kawano, Y and Nagamine, M and Hayashi, T and Inoue, K},
title = {Unexpected genomic features of high current density-producing Geobacter sulfurreducens strain YM18.},
journal = {FEMS microbiology letters},
volume = {368},
number = {17},
pages = {},
doi = {10.1093/femsle/fnab119},
pmid = {34472610},
issn = {1574-6968},
abstract = {Geobacter sulfurreducens produces high current densities and it has been used as a model organism for extracellular electron transfer studies. Nine G. sulfurreducens strains were isolated from biofilms formed on an anode poised at -0.2 V (vs SHE) in a bioelectrochemical system in which river sediment was used as an inoculum. The maximum current density of an isolate, strain YM18 (9.29 A/m2), was higher than that of the strain PCA (5.72 A/m2), the type strain of G. sulfurreducens, and comparable to strain KN400 (8.38 A/m2), which is another high current-producing strain of G. sulfurreducens. Genomic comparison of strains PCA, KN400 and YM18 revealed that omcB, xapD, spc and ompJ, which are known to be important genes for iron reduction and current production in PCA, were not present in YM18. In the PCA and KN400 genomes, two and one region(s) encoding CRISPR/Cas systems were identified, respectively, but they were missing in the YM18 genome. These results indicate that there is genetic variation in the key components involved in extracellular electron transfer among G. sulfurreducens strains.},
}

@article {pmid34472225,
year = {2021},
author = {Cui, Y and Liu, ZL and Li, CC and Wei, XM and Lin, YJ and You, L and Zhu, ZD and Deng, HM and Feng, QL and Huang, YP and Xiang, H},
title = {Role of juvenile hormone receptor Methoprene-tolerant 1 in silkworm larval brain development and domestication.},
journal = {Zoological research},
volume = {42},
number = {5},
pages = {637-649},
pmid = {34472225},
issn = {2095-8137},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; Bombyx/growth & development/*metabolism ; Brain/growth & development/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation, Developmental ; Genotype ; Insect Proteins/genetics/*metabolism ; Integumentary System/physiology ; Juvenile Hormones/*metabolism ; Larva/growth & development/metabolism ; Phylogeny ; Protein Conformation ; },
abstract = {The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 (Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.},
}

Amino Acid Sequence
Animals
Base Sequence
Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism
Bombyx/growth & development/*metabolism
Brain/growth & development/metabolism
CRISPR-Cas Systems
Gene Deletion
Gene Expression Regulation, Developmental
Genotype
Insect Proteins/genetics/*metabolism
Integumentary System/physiology
Juvenile Hormones/*metabolism
Larva/growth & development/metabolism
Phylogeny
Protein Conformation

@article {pmid34471706,
year = {2021},
author = {O'Leary, JK and Sleator, RD and Lucey, B},
title = {Cryptosporidium spp. diagnosis and research in the 21st century.},
journal = {Food and waterborne parasitology},
volume = {24},
number = {},
pages = {e00131},
pmid = {34471706},
issn = {2405-6766},
abstract = {The protozoan parasite Cryptosporidium has emerged as a leading cause of diarrhoeal illness worldwide, posing a significant threat to young children and immunocompromised patients. While endemic in the vast majority of developing countries, Cryptosporidium also has the potential to cause waterborne epidemics and large scale outbreaks in both developing and developed nations. Anthroponontic and zoonotic transmission routes are well defined, with the ingestion of faecally contaminated food and water supplies a common source of infection. Microscopy, the current diagnostic mainstay, is considered by many to be suboptimal. This has prompted a shift towards alternative diagnostic techniques in the advent of the molecular era. Molecular methods, particularly PCR, are gaining traction in a diagnostic capacity over microscopy in the diagnosis of cryptosporidiosis, given the laborious and often tedious nature of the latter. Until now, developments in the field of Cryptosporidium detection and research have been somewhat hampered by the intractable nature of this parasite. However, recent advances in the field have taken the tentative first steps towards bringing Cryptosporidium research into the 21st century. Herein, we provide a review of these advances.},
}

@article {pmid34471126,
year = {2021},
author = {Tong, Y and Jørgensen, TS and Whitford, CM and Weber, T and Lee, SY},
title = {A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5206},
pmid = {34471126},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial ; Escherichia coli/*genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Bacterial ; Plasmids ; RNA, Guide/genetics ; },
abstract = {CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA, Bacterial
Escherichia coli/*genetics
Gene Editing/*methods
Genetic Engineering/*methods
Genome, Bacterial
Plasmids
RNA, Guide/genetics

@article {pmid34466691,
year = {2021},
author = {Chen, J and Zhuang, X and Zheng, J and Yang, R and Wu, F and Zhang, A and Fang, B},
title = {Aptamer-based cell-free detection system to detect target protein.},
journal = {Synthetic and systems biotechnology},
volume = {6},
number = {3},
pages = {209-215},
pmid = {34466691},
issn = {2405-805X},
abstract = {Biomarkers of disease, especially protein, show great potential for diagnosis and prognosis. For detecting a certain protein, a binding assay implementing antibodies is commonly performed. However, antibodies are not thermally stable and may cause false-positive when the sample composition is complicated. In recent years, a functional nucleic acid named aptamer has been used in many biochemical analysis cases, which is commonly selected from random sequence libraries by using the systematic evolution of ligands by exponential enrichment (SELEX) techniques. Compared to antibodies, the aptamer is more thermal stable, easier to be modified, conjugated, and amplified. Herein, an Aptamer-Based Cell-free Detection (ABCD) system was proposed to detect target protein, using epithelial cell adhesion molecule (EpCAM) as an example. We combined the robustness of aptamer in binding specificity with the signal amplification ability of CRISPR-Cas12a's trans-cleavage activity in the ABCD system. We also demonstrated that the ABCD system could work well to detect target protein in a relatively low limit of detection (50-100 nM), which lay a foundation for the development of portable detection devices. This work highlights the superiority of the ABCD system in detecting target protein with low abundance and offers new enlightenment for future design and development.},
}

@article {pmid34465535,
year = {2021},
author = {Jung, C and Till, B},
title = {Mutagenesis and genome editing in crop improvement: perspectives for the global regulatory landscape.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2021.08.002},
pmid = {34465535},
issn = {1878-4372},
abstract = {Plant breeding depends on broad genetic variation. New allelic variation can be produced by targeted or random mutagenesis. Seemingly, random mutagenesis is outdated because clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology is much more precise and potentially faster. Unfortunately, genome editing is not accessible to breeders in many countries due to legal constraints. Therefore, random mutagenesis remains a vital method to create new allelic variation. Mutant offspring, however, suffer from a heavy mutation load, and application in polyploid crops is limited because multiple mutations are typically required. Exploiting random mutations became more efficient due to recent technological advancements, such as sequence-based mutant screening and genomic background selection. In this review, random and targeted mutagenesis will be compared, highlighting the legal situation.},
}

@article {pmid34465216,
year = {2021},
author = {Ongenae, V and Briegel, A and Claessen, D},
title = {Cell wall deficiency as an escape mechanism from phage infection.},
journal = {Open biology},
volume = {11},
number = {9},
pages = {210199},
pmid = {34465216},
issn = {2046-2441},
abstract = {The cell wall plays a central role in protecting bacteria from some environmental stresses, but not against all. In fact, in some cases, an elaborate cell envelope may even render the cell more vulnerable. For example, it contains molecules or complexes that bacteriophages recognize as the first step of host invasion, such as proteins and sugars, or cell appendages such as pili or flagella. In order to counteract phages, bacteria have evolved multiple escape mechanisms, such as restriction-modification, abortive infection, CRISPR/Cas systems or phage inhibitors. In this perspective review, we present the hypothesis that bacteria may have additional means to escape phage attack. Some bacteria are known to be able to shed their cell wall in response to environmental stresses, yielding cells that transiently lack a cell wall. In this wall-less state, the bacteria may be temporarily protected against phages, since they lack the essential entities that are necessary for phage binding and infection. Given that cell wall deficiency can be triggered by clinically administered antibiotics, phage escape could be an unwanted consequence that limits the use of phage therapy for treating stubborn infections.},
}

@article {pmid34461727,
year = {2021},
author = {Shen, H and Qileng, A and Yang, H and Liang, H and Zhu, H and Liu, Y and Lei, H and Liu, W},
title = {"Dual-Signal-On" Integrated-Type Biosensor for Portable Detection of miRNA: Cas12a-Induced Photoelectrochemistry and Fluorescence Strategy.},
journal = {Analytical chemistry},
volume = {93},
number = {34},
pages = {11816-11825},
doi = {10.1021/acs.analchem.1c02395},
pmid = {34461727},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; DNA ; Horseradish Peroxidase ; *MicroRNAs/genetics ; },
abstract = {The abnormal expression of microRNA (miRNA) can affect the RNA transcription and protein translation, leading to tumor progression and metastasis. Currently, the accurate detection of aberrant expression of miRNA, particularly using a portable detection system, remains a great challenge. Herein, a novel dual-mode biosensor with high sensitivity and robustness for miR-21 detection was developed based on the cis-cleavage and trans-cleavage activities of Cas12a. miRNA can be combined with hairpin DNA-horseradish peroxidase anchored on a CdS/g-C3N4/B-TiO2 photoelectrode, thus the nonenzymatic amplification was triggered to form numerous HRP-modified double-stranded DNA (HRP-dsDNA). Then, HRP-dsDNA can be specifically recognized and efficiently cis-cleaved by Cas12a nucleases to detach HRP from the substrate, while the remaining HRP on HRP-dsDNA can catalyze 4-chloro-1-naphthol (4-CN) to form biocatalytic precipitation (BCP) on the surface of the photoelectrode, and thus the photocurrent can be changed. Meanwhile, the trans-cleavage ability of Cas12a was activated, and nonspecifically degrade the FQ-reporter and a significant fluorescence signal can be generated. Such two different kinds of signals with independent transmission paths can mutually support to improve the performance of the detection platform. Besides, a portable device was constructed for the point-of-care (POC) detection of miR-21. Moreover, the dual-mode detection platform can be easily expanded for the specific detection of other types of biomarkers by changing the sequence of hairpin DNA, thereby promoting the establishment of POC detection for early cancer diagnosis.},
}

*Biosensing Techniques
CRISPR-Cas Systems
DNA
Horseradish Peroxidase
*MicroRNAs/genetics

@article {pmid34461102,
year = {2021},
author = {Pollecker, K and Sylvester, M and Voos, W},
title = {Proteomic analysis demonstrates the role of the quality control protease LONP1 in mitochondrial protein aggregation.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {101134},
doi = {10.1016/j.jbc.2021.101134},
pmid = {34461102},
issn = {1083-351X},
abstract = {The mitochondrial matrix protease LONP1 is an essential part of the organellar protein quality control system. LONP1 has been shown to be involved in respiration control and apoptosis. Furthermore, a reduction in LONP1 level correlates with ageing. Up to now, the effects of a LONP1 defect were mostly studied by utilizing transient, siRNA-mediated knockdown approaches. We generated a new cellular model system for studying the impact of LONP1 on mitochondrial protein homeostasis by a CRISPR/Cas-mediated genetic knockdown (gKD). These cells show a stable reduction of LONP1 along with a mild phenotype characterized by absent morphological differences and only small negative effects on mitochondrial functions under normal culture conditions. To assess the consequences of a permanent LONP1 depletion on the mitochondrial proteome, we analyzed the alterations of protein levels by quantitative mass spectrometry, demonstrating small adaptive changes, in particular with respect to mitochondrial protein biogenesis. In an additional proteomic analysis, we determined the temperature-dependent aggregation behavior of mitochondrial proteins and its dependence on a reduction of LONP1 activity, demonstrating the important role of the protease for mitochondrial protein homeostasis in mammalian cells. We identified a significant number of mitochondrial proteins that are affected by LONP1 activity especially with respect to their stress-induced solubility. Taken together, our results suggest a very good applicability of the LONP1 gKD cell line as a model system for human ageing processes.},
}

@article {pmid34456559,
year = {2021},
author = {Asmamaw, M and Zawdie, B},
title = {Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing.},
journal = {Biologics : targets & therapy},
volume = {15},
number = {},
pages = {353-361},
pmid = {34456559},
issn = {1177-5475},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines. Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The mechanism of CRISPR/Cas-9 genome editing contains three steps, recognition, cleavage, and repair. The designed sgRNA recognizes the target sequence in the gene of interest through a complementary base pair. While the Cas-9 nuclease makes double-stranded breaks at a site 3 base pair upstream to protospacer adjacent motif, then the double-stranded break is repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. The CRISPR/Cas-9 genome-editing tool has a wide number of applications in many areas including medicine, agriculture, and biotechnology. In agriculture, it could help in the design of new grains to improve their nutritional value. In medicine, it is being investigated for cancers, HIV, and gene therapy such as sickle cell disease, cystic fibrosis, and Duchenne muscular dystrophy. The technology is also being utilized in the regulation of specific genes through the advanced modification of Cas-9 protein. However, immunogenicity, effective delivery systems, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications. Although CRISPR/Cas-9 becomes a new era in molecular biology and has countless roles ranging from basic molecular researches to clinical applications, there are still challenges to rub in the practical applications and various improvements are needed to overcome obstacles.},
}

@article {pmid34456394,
year = {2021},
author = {Novikov, RV and Gribkova, AK and Kacher, JG and Zaytsev, PA and Armeev, GA and Gluhov, GS and Shaytan, AK},
title = {Design of Nucleic Acid Biosensors Based on CRISPR/Cas Systems and Reporter Split Proteins.},
journal = {Moscow University biological sciences bulletin},
volume = {76},
number = {2},
pages = {52-58},
pmid = {34456394},
issn = {0096-3925},
abstract = {Highly sensitive, specific, rapid, and easy-to-use diagnostic methods for the detection of nucleic acids of pathogens are required for the diagnosis of many human, animal, and plant diseases and environmental monitoring. The approaches based on the use of the natural ability of bacterial CRISPR/Cas9 systems to recognize DNA sequences with a high specificity under isothermal conditions are an alternative to the polymerase chain reaction method, which requires expensive laboratory equipment. The development of the methods for signal registration with the formation of a DNA/RNA/Cas9 protein complex is a separate bioengineering task. In this work, a design was developed and the applicability of a biosensor system based on the binding of two dCas9 proteins with target DNA sequences (without their cutting) and detection of their colocalization using reporter systems based on split enzymes was studied. Using the methods of molecular modeling, possible mutual positions of two dCas9 proteins at a detectable locus of genomic DNA, allowing the split enzyme domains attached to them to interact in an optimal way, were determined. The optimal distances on DNA between binding sites of dCas9 proteins in different orientations were determined, and the dependence of the complex structure on the distance between the binding sites of dCas9 proteins was modeled. Using the methods of bioinformatics, the genomes of a number of viruses (including SARS-CoV-2) were analyzed, and the presence of genomic loci unique to the species, allowing the possibility of landing pairs of dCas9 proteins in optimal positions, was demonstrated. The possibility of a combined use of dCas9 proteins from different bacteria to expand the spectrum of detected loci was analyzed. The results of the work indicate a fundamental possibility of the creation of highly specific nucleic acid biosensors based on a combination of CRISPR/Cas9 technologies and split enzymes.},
}

@article {pmid34455224,
year = {2021},
author = {Kim, H and Lee, S and Yoon, J and Song, J and Park, HG},
title = {CRISPR/Cas12a collateral cleavage activity for simple and rapid detection of protein/small molecule interaction.},
journal = {Biosensors & bioelectronics},
volume = {194},
number = {},
pages = {113587},
doi = {10.1016/j.bios.2021.113587},
pmid = {34455224},
issn = {1873-4235},
abstract = {To realize the full potential of the CRISPR/Cas system and expand its applicability up to the detection of molecular interactions, we herein describe a novel method to identify protein/small molecule interactions by utilizing the CRISPR/Cas12a collateral cleavage activity. This technique employs a single-stranded activator DNA modified with a specific small molecule, which would switch on the CRISPR/Cas12a collateral cleavage activity upon binding to crRNA within the CRISPR/Cas12a system. When the target protein binds to the small molecule on the activator DNA, the bound protein sterically hinders the access of the activator DNA to crRNA, thereby promoting less collateral cleavage activity of CRISPR/Cas12a. As a consequence, fewer reporter probes nearby are cleaved to produce accordingly reduced fluorescence signals in response to target protein. Based on this unique design principle, the two model protein/small molecule interactions, streptavidin/biotin and anti-digoxigenin/digoxigenin, were successfully determined down to 0.03 nM and 0.09 nM, respectively, with a fast and simple detection workflow (11 min). The practical applicability of this method was also verified by reliably detecting target streptavidin spiked in heterogeneous human serum. This work would provide great insight to construct novel strategies to identify protein/small molecule interaction by making the most of the CRISPR/Cas12a system beyond its superior capabilities in genome editing and molecular diagnostics.},
}

@article {pmid34454311,
year = {2021},
author = {Huang, M and Liu, M and Huang, L and Wang, M and Jia, R and Zhu, D and Chen, S and Zhao, X and Zhang, S and Gao, Q and Zhang, L and Cheng, A},
title = {The activation and limitation of the bacterial natural transformation system: The function in genome evolution and stability.},
journal = {Microbiological research},
volume = {252},
number = {},
pages = {126856},
doi = {10.1016/j.micres.2021.126856},
pmid = {34454311},
issn = {1618-0623},
abstract = {Bacteria can take up exogenous naked DNA and integrate it into their genomes, which has been regarded as a main contributor to bacterial evolution. The competent status of bacteria is influenced by environmental cues and by the immune systems of bacteria. Here, we review recent advances in understanding the working mechanisms underlying activation of the natural transformation system and limitations thereof. Environmental stresses including the presence of antimicrobials can activate the natural transformation system. However, bacterial enzymes (nucleases), non-coding RNAs, specific DNA sequences, the restriction-modification (R-M) systems, CRISPR-Cas systems and prokaryotic Argonaute proteins (Agos) are have been found to be involved in the limitation of the natural transformation system. Together, this review represents an opportunity to gain insight into bacterial genome stability and evolution.},
}

@article {pmid34454111,
year = {2021},
author = {Ahmed, MZ and Badani, P and Reddy, R and Mishra, G},
title = {CRISPR/Cas Advancement in Molecular Diagnostics and Signal Readout Approaches.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmoldx.2021.07.025},
pmid = {34454111},
issn = {1943-7811},
abstract = {Discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has greatly improved our gene editing technology. Their applications in the area of diagnostic innovation are gaining much attention. The key characteristics of CRISPR/Cas system that allowed its extensive exploitation in the detection platform are their programmable and highly selective target recognition scheme. We present herein the major three Cas effectors (Cas9, Cas12, and Cas13) and their significance in various detection assays. The CRISPR/Cas detection strategies, based on their target hybridization, cleavage activity, sensor capabilities, and signal readout methods, are discussed. We also highlighted some of the recent progressions, challenges, and improvement strategies of CRISPR/Cas technology and their biosensing detection platforms toward the development of simple, sensitive, and portable point-of-care diagnostic devices.},
}

@article {pmid34453943,
year = {2021},
author = {Luthra, R and Kaur, S and Bhandari, K},
title = {Applications of CRISPR as a potential therapeutic.},
journal = {Life sciences},
volume = {284},
number = {},
pages = {119908},
doi = {10.1016/j.lfs.2021.119908},
pmid = {34453943},
issn = {1879-0631},
abstract = {Genetic disorders and congenital abnormalities are present in 2-5% of births all over the world and can cause up to 50% of all early childhood deaths. The establishment of sophisticated and controlled techniques for customizing DNA manipulation is significant for the therapeutic role in such disorders and further research on them. One such technique is CRISPR that is significant towards optimizing genome editing and therapies, metabolic fluxes as well as artificial genetic systems. CRISPR-Cas9 is a molecular appliance that is applied in the areas of genetic and protein engineering. The CRISPR-CAS system is an integral element of prokaryotic adaptive immunity that allows prokaryotic cells to identify and kill any foreign DNA. The Gene editing property of CRISPR finds various applications like diagnostics and therapeutics in cancer, neurodegenerative disorders, genetic diseases, blindness, etc. This review discusses applications of CRISPR as a therapeutic in various disorders including several genetic diseases (including sickle cell anemia, blindness, thalassemia, cystic fibrosis, hereditary tyrosinemia type I, duchenne muscular dystrophy, mitochondrial disorders), Cancer, Huntington's disease and viral infections (like HIV, COVID, etc.) along with the prospects concerning them. CRISPR-based therapy is also being researched and defined for COVID-19. The related mechanism of CRISPR has been discussed alongside highlighting challenges involved in therapeutic applications of CRISPR.},
}

@article {pmid34450043,
year = {2021},
author = {Xiao, R and Wang, S and Han, R and Li, Z and Gabel, C and Mukherjee, IA and Chang, L},
title = {Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2021.07.043},
pmid = {34450043},
issn = {1097-4164},
abstract = {The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.},
}

@article {pmid34446818,
year = {2021},
author = {Uribe, RV and Rathmer, C and Jahn, LJ and Ellabaan, MMH and Li, SS and Sommer, MOA},
title = {Bacterial resistance to CRISPR-Cas antimicrobials.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {17267},
pmid = {34446818},
issn = {2045-2322},
support = {NNF20CC0035580//Novo Nordisk Fonden/ ; R140-2013-13496//Lundbeckfonden/ ; No 642738//H2020 Marie Skłodowska-Curie Actions/ ; ALTF 137-2018//European Molecular Biology Laboratory/ ; NHMRC; 1166180//Australian National Health and Medical Research Council/ ; },
abstract = {In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.},
}

@article {pmid34446442,
year = {2021},
author = {van Beljouw, SPB and Haagsma, AC and Rodríguez-Molina, A and van den Berg, DF and Vink, JNA and Brouns, SJJ},
title = {The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6561},
pages = {1349-1353},
doi = {10.1126/science.abk2718},
pmid = {34446442},
issn = {1095-9203},
mesh = {Bacteria/*enzymology/*genetics ; Bacterial Proteins/chemistry/*metabolism ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; Caspases/chemistry/metabolism ; Endoribonucleases/chemistry/genetics/*metabolism ; Interspersed Repetitive Sequences ; Peptide Hydrolases/chemistry/*metabolism ; Protein Domains ; RNA, Bacterial/metabolism ; RNA, Viral/metabolism ; Substrate Specificity ; },
abstract = {[Figure: see text].},
}

Bacteria/*enzymology/*genetics
Bacterial Proteins/chemistry/*metabolism
CRISPR-Associated Proteins/chemistry/genetics/*metabolism
*CRISPR-Cas Systems
Caspases/chemistry/metabolism
Endoribonucleases/chemistry/genetics/*metabolism
Interspersed Repetitive Sequences
Peptide Hydrolases/chemistry/*metabolism
Protein Domains
RNA, Bacterial/metabolism
RNA, Viral/metabolism
Substrate Specificity

@article {pmid34446204,
year = {2021},
author = {Han, W and Tang, F and Zhong, Y and Zhang, J and Liu, Z},
title = {Identification of yellow gene family and functional analysis of Spodoptera frugiperda yellow-y by CRISPR/Cas9.},
journal = {Pesticide biochemistry and physiology},
volume = {178},
number = {},
pages = {104937},
doi = {10.1016/j.pestbp.2021.104937},
pmid = {34446204},
issn = {1095-9939},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Insecticide Resistance ; Larva/genetics ; *Moths ; Spodoptera/genetics ; },
abstract = {For a devastating agricultural pest, functional genomics promotes the finding of novel technology to control Spodoptera frugiperda, such as the genetics-based strategies. In the present study, 11 yellow genes were identified in Spodoptera frugiperda. The transcriptome analysis showed the tissue-specific expression of part yellow genes, which suggested the importance of yellow genes in some biological processes in S. frugiperda, such as pigmentation. Among these yellow genes, the expression profiles of yellow-y gene showed that it was expressed in all life stages. In order to realize the further study of yellow-y, we employed CRISPR/Cas9 system to knock out this gene. Following knock out, diverse phenotypes were observed, such as color changes in both larvae and adults. Different from the wild-type larvae and adults, G0 mutants were yellowed since hatching. However, no color difference was observed with the pupal cuticle between the wild-type and mutant pupae before the 8th day. On the basis of the single-pair strategy of G0 generation, the yellow-y gene was proved to be a recessive gene. The G1 yellowish larvae with biallelic mutations displayed a relatively longer development period than wild-type, and often generated abnormal pupae and moths. The deletion of yellow-y also resulted in a decline in the fecundity. The results revealed that yellow-y gene was important for S. frugiperda pigmentation, as well as in its development and reproduction. Besides, the present study set up a standard procedure to knock out genes in S. frugiperda, which could be helpful for our understanding some key molecular processes, such as functional roles of detoxification genes as insecticide resistance mechanisms or modes of action of insecticides to facilitate the management of this insect pest.},
}

Animals
*CRISPR-Cas Systems/genetics
Insecticide Resistance
Larva/genetics
*Moths
Spodoptera/genetics

@article {pmid34446084,
year = {2021},
author = {Ghaffari, S and Khalili, N and Rezaei, N},
title = {CRISPR/Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy.},
journal = {Journal of experimental & clinical cancer research : CR},
volume = {40},
number = {1},
pages = {269},
pmid = {34446084},
issn = {1756-9966},
abstract = {Cancer immunotherapy has gained attention as the supreme therapeutic modality for the treatment of various malignancies. Adoptive T-cell therapy (ACT) is one of the most distinctive modalities of this therapeutic approach, which seeks to harness the potential of combating cancer cells by using autologous or allogenic tumor-specific T-cells. However, a plethora of circumstances must be optimized to produce functional, durable, and efficient T-cells. Recently, the potential of ACT has been further realized by the introduction of novel gene-editing platforms such as the CRISPR/Cas9 system; this technique has been utilized to create T-cells furnished with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR) that have precise tumor antigen recognition, minimal side effects and treatment-related toxicities, robust proliferation and cytotoxicity, and nominal exhaustion. Here, we aim to review and categorize the recent breakthroughs of genetically modified TCR/CAR T-cells through CRISPR/Cas9 technology and address the pearls and pitfalls of each method. In addition, we investigate the latest ongoing clinical trials that are applying CRISPR-associated TCR/CAR T-cells for the treatment of cancers.},
}

@article {pmid34445589,
year = {2021},
author = {Shaw, AM and Qasem, A and Naser, SA},
title = {Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445589},
issn = {1422-0067},
support = {2014-2020//Florida Legislative Grant/ ; },
mesh = {Arthritis, Rheumatoid/genetics ; *CRISPR-Cas Systems ; Crohn Disease/genetics ; Gene Expression Regulation, Leukemic/*drug effects ; Genetic Predisposition to Disease ; Humans ; Jurkat Cells ; Leukemia, T-Cell/drug therapy/genetics/*pathology ; Lymphocyte Activation ; *Polymorphism, Single Nucleotide ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/antagonists & inhibitors/*genetics/metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 22/antagonists & inhibitors/*genetics/metabolism ; Spermidine/*pharmacology ; },
abstract = {Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 μM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.},
}

Arthritis, Rheumatoid/genetics
*CRISPR-Cas Systems
Crohn Disease/genetics
Gene Expression Regulation, Leukemic/*drug effects
Genetic Predisposition to Disease
Humans
Jurkat Cells
Leukemia, T-Cell/drug therapy/genetics/*pathology
Lymphocyte Activation
*Polymorphism, Single Nucleotide
Protein Tyrosine Phosphatase, Non-Receptor Type 2/antagonists & inhibitors/*genetics/metabolism
Protein Tyrosine Phosphatase, Non-Receptor Type 22/antagonists & inhibitors/*genetics/metabolism
Spermidine/*pharmacology

@article {pmid34445569,
year = {2021},
author = {Sarkar, H and Toms, M and Moosajee, M},
title = {Involvement of Oxidative and Endoplasmic Reticulum Stress in RDH12-Related Retinopathies.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445569},
issn = {1422-0067},
support = {205174/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; //Retina UK/ ; },
mesh = {Alcohol Oxidoreductases/antagonists & inhibitors/genetics/*metabolism ; Animals ; *Apoptosis ; Autophagy ; CRISPR-Cas Systems ; *Endoplasmic Reticulum Stress ; HEK293 Cells ; Humans ; *Mutation ; *Oxidative Stress ; Retinal Diseases/etiology/metabolism/*pathology ; Zebrafish ; },
abstract = {Retinol dehydrogenase 12 (RDH12) is expressed in photoreceptor inner segments and catalyses the reduction of all-trans retinal (atRAL) to all-trans retinol (atROL), as part of the visual cycle. Mutations in RDH12 are primarily associated with autosomal recessive Leber congenital amaurosis. To further our understanding of the disease mechanisms, HEK-293 cell lines expressing wildtype (WT) and mutant RDH12 were created. The WT cells afforded protection from atRAL-induced toxicity and oxidative stress. Mutant RDH12 cells displayed reduced protein expression and activity, with an inability to protect cells from atRAL toxicity, inducing oxidative and endoplasmic reticulum (ER) stress, with upregulation of sXBP1, CHOP, and ATF4. Pregabalin, a retinal scavenger, attenuated atRAL-induced ER stress in the mutant RDH12 cell lines. A zebrafish rdh12 mutant model (rdh12u533 c.17_23del; p.(Val6AlafsTer5)) was generated through CRISPR-Cas9 gene editing. Mutant fish showed disrupted phagocytosis through transmission electron microscopy, with increased phagosome size at 12 months post-fertilisation. Rhodopsin mislocalisation and reduced expression of atg12 and sod2 indicated early signs of a rod-predominant degeneration. A lack of functional RDH12 results in ER and oxidative stress representing key pathways to be targeted for potential therapeutics.},
}

Alcohol Oxidoreductases/antagonists & inhibitors/genetics/*metabolism
Animals
*Apoptosis
Autophagy
CRISPR-Cas Systems
*Endoplasmic Reticulum Stress
HEK293 Cells
Humans
*Mutation
*Oxidative Stress
Retinal Diseases/etiology/metabolism/*pathology
Zebrafish

@article {pmid34445458,
year = {2021},
author = {Khusnutdinov, E and Sukhareva, A and Panfilova, M and Mikhaylova, E},
title = {Anthocyanin Biosynthesis Genes as Model Genes for Genome Editing in Plants.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445458},
issn = {1422-0067},
support = {20-74-10053//Russian Science Foundation/ ; },
mesh = {*Anthocyanins/biosynthesis/genetics ; *Gene Editing ; *Genes, Plant ; *Models, Genetic ; *Plants/genetics/metabolism ; },
abstract = {CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as targets. Anthocyanin pigmentation is one of the most easily identified traits, that does not require any additional treatment. It is also associated with stress resistance, therefore plants with edited anthocyanin genes might be of interest for agriculture. Phenotypic effect of CRISPR/Cas editing of PAP1 and its homologs, DFR, F3H and F3'H genes have been confirmed in several distinct plant species. DFR appears to be a key structural gene of anthocyanin biosynthesis, controlled by various transcription factors. There are still many promising potential model genes that have not been edited yet. Some of them, such as Delila, MYB60, HAT1, UGT79B2, UGT79B3 and miR156, have been shown to regulate drought tolerance in addition to anthocyanin biosynthesis. Genes, also involved in trichome development, such as TTG1, GLABRA2, MYBL2 and CPC, can provide increased visibility. In this review successful events of CRISPR/Cas editing of anthocyanin genes are summarized, and new model genes are proposed. It can be useful for molecular biologists and genetic engineers, crop scientists, plant genetics and physiologists.},
}

*Anthocyanins/biosynthesis/genetics
*Gene Editing
*Genes, Plant
*Models, Genetic
*Plants/genetics/metabolism