@article {pmid34687393,
year = {2021},
author = {Dilip Kumar, S and Aashabharathi, M and KarthigaDevi, G and Subbaiya, R and Saravanan, M},
title = {Insights of CRISPR-Cas systems in stem cells: progress in regenerative medicine.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {34687393},
issn = {1573-4978},
abstract = {Regenerative medicine, a therapeutic approach using stem cells, aims to rejuvenate and restore the normalized function of the cells, tissues, and organs that are injured, malfunctioning, and afflicted. This influential technology reaches its zenith when it is integrated with the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) technology of genome editing. This tool acts as a programmable restriction enzyme system, which targets DNA as well as RNA and gets redeployed for the customization of DNA/RNA sequences. The dynamic behaviour of nuclear manipulation and transcriptional regulation by CRISPR-Cas technology renders it with numerous employment in the field of biologics and research. Here, the possible impact of the commonly practiced CRISPR-Cas systems in regenerative medicines is being reviewed. Primarily, the discussion of the working mechanism of this system and the fate of stem cells will be scrutinized. A detailed description of the CRISPR based regenerative therapeutic approaches for a horde of diseases like genetic disorders, neural diseases, and blood-related diseases is elucidated.},
}

@article {pmid34686666,
year = {2021},
author = {Buddhachat, K and Paenkaew, S and Sripairoj, N and Gupta, YM and Pradit, W and Chomdej, S},
title = {Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {20888},
pmid = {34686666},
issn = {2045-2322},
abstract = {Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen's DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, "Bar-cas12a" for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM's gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.},
}

@article {pmid34685392,
year = {2021},
author = {Alok, A and Chauhan, H and Upadhyay, SK and Pandey, A and Kumar, J and Singh, K},
title = {Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages.},
journal = {Life (Basel, Switzerland)},
volume = {11},
number = {10},
pages = {},
doi = {10.3390/life11101021},
pmid = {34685392},
issn = {2075-1729},
abstract = {CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.},
}

@article {pmid34684206,
year = {2021},
author = {Magro, G and Calistri, A and Parolin, C},
title = {Targeting and Understanding HIV Latency: The CRISPR System against the Provirus.},
journal = {Pathogens (Basel, Switzerland)},
volume = {10},
number = {10},
pages = {},
doi = {10.3390/pathogens10101257},
pmid = {34684206},
issn = {2076-0817},
support = {PARO_FINA20_01//University of Padua/ ; },
abstract = {The presence of latently infected cells and reservoirs in HIV-1 infected patients constitutes a significant obstacle to achieve a definitive cure. Despite the efforts dedicated to solve these issues, the mechanisms underlying viral latency are still under study. Thus, on the one hand, new strategies are needed to elucidate which factors are involved in latency establishment and maintenance. On the other hand, innovative therapeutic approaches aimed at eradicating HIV infection are explored. In this context, advances of the versatile CRISPR-Cas gene editing technology are extremely promising, by providing, among other advantages, the possibility to target the HIV-1 genome once integrated into cellular DNA (provirus) and/or host-specific genes involved in virus infection/latency. This system, up to now, has been employed with success in numerous in vitro and in vivo studies, highlighting its increasing significance in the field. In this review, we focus on the progresses made in the use of different CRISPR-Cas strategies to target the HIV-1 provirus, and we then discuss recent advancements in the use of CRISPR screens to elucidate the role of host-specific factors in viral latency.},
}

@article {pmid34681959,
year = {2021},
author = {Naeem, M and Hoque, MZ and Ovais, M and Basheer, C and Ahmad, I},
title = {Stimulus-Responsive Smart Nanoparticles-Based CRISPR-Cas Delivery for Therapeutic Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {20},
pages = {},
doi = {10.3390/ijms222011300},
pmid = {34681959},
issn = {1422-0067},
abstract = {The innovative research in genome editing domains such as CRISPR-Cas technology has enabled genetic engineers to manipulate the genomes of living organisms effectively in order to develop the next generation of therapeutic tools. This technique has started the new era of "genome surgery". Despite these advances, the barriers of CRISPR-Cas9 techniques in clinical applications include efficient delivery of CRISPR/Cas9 and risk of off-target effects. Various types of viral and non-viral vectors are designed to deliver the CRISPR/Cas9 machinery into the desired cell. These methods still suffer difficulties such as immune response, lack of specificity, and efficiency. The extracellular and intracellular environments of cells and tissues differ in pH, redox species, enzyme activity, and light sensitivity. Recently, smart nanoparticles have been synthesized for CRISPR/Cas9 delivery to cells based on endogenous (pH, enzyme, redox specie, ATP) and exogenous (magnetic, ultrasound, temperature, light) stimulus signals. These methodologies can leverage genome editing through biological signals found within disease cells with less off-target effects. Here, we review the recent advances in stimulus-based smart nanoparticles to deliver the CRISPR/Cas9 machinery into the desired cell. This review article will provide extensive information to cautiously utilize smart nanoparticles for basic biomedical applications and therapeutic genome editing.},
}

@article {pmid34681568,
year = {2021},
author = {Molina-Risco, M and Ibarra, O and Faion-Molina, M and Kim, B and Septiningsih, EM and Thomson, MJ},
title = {Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio.},
journal = {International journal of molecular sciences},
volume = {22},
number = {20},
pages = {},
doi = {10.3390/ijms222010909},
pmid = {34681568},
issn = {1422-0067},
support = {2020-67013-31811//United States Department of Agriculture/ ; 2018 X-Grant//Texas A&M University/ ; Research funds//Texas A&M AgriLife Research/ ; },
abstract = {Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.},
}

@article {pmid34680899,
year = {2021},
author = {Lu, W and Pei, Z and Zang, M and Lee, YK and Zhao, J and Chen, W and Wang, H and Zhang, H},
title = {Comparative Genomic Analysis of Bifidobacterium bifidum Strains Isolated from Different Niches.},
journal = {Genes},
volume = {12},
number = {10},
pages = {},
doi = {10.3390/genes12101504},
pmid = {34680899},
issn = {2073-4425},
abstract = {The potential probiotic benefits of Bifidobacterium bifidum have received increasing attention recently. We used comparative genomic analysis to explore the differences in the genome and the physiological characteristics of B. bifidum isolated from the fecal samples of Chinese adults and infants. The relationships between genotypes and phenotypes were analyzed to assess the effects of isolation sources on the genetic variation of B. bifidum. The phylogenetic tree results indicated that the phylogeny of B. bifidum may be related to the geographical features of its isolation source. B. bifidum was found to have an open pan-genome and a conserved core genome. The genetic diversity of B. bifidum is mainly reflected in carbohydrate metabolism- and immune/competition-related factors, such as the glycoside hydrolase gene family, bacteriocin operons, antibiotic resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Additionally, the type III A CRISPR-Cas system was discovered in B. bifidum for the first time. B. bifidum strains exhibited niche-specific characteristics, and the results of this study provide an improved understanding of the genetics of this species.},
}

@article {pmid34588447,
year = {2021},
author = {Sapozhnikov, DM and Szyf, M},
title = {Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5711},
pmid = {34588447},
issn = {2041-1723},
support = {PJT-159583//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; CpG Islands/genetics ; *DNA Demethylation ; DNA Methylation ; DNA Modification Methylases/genetics/*metabolism ; *Epigenesis, Genetic ; Fragile X Mental Retardation Protein/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Mice ; Mixed Function Oxygenases/genetics/*metabolism ; NIH 3T3 Cells ; Promoter Regions, Genetic/genetics ; Proto-Oncogene Proteins/genetics/*metabolism ; RNA, Guide/metabolism ; Serpins/genetics ; },
abstract = {Despite four decades of research to support the association between DNA methylation and gene expression, the causality of this relationship remains unresolved. Here, we reaffirm that experimental confounds preclude resolution of this question with existing strategies, including recently developed CRISPR/dCas9 and TET-based epigenetic editors. Instead, we demonstrate a highly effective method using only nuclease-dead Cas9 and guide RNA to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzyme, thereby enabling the examination of the role of DNA demethylation per se in living cells, with no evidence of off-target activity. Using this method, we probe a small number of inducible promoters and find the effect of DNA demethylation to be small, while demethylation of CpG-rich FMR1 produces larger changes in gene expression. This method could be used to reveal the extent and nature of the contribution of DNA methylation to gene regulation.},
}

Animals
CRISPR-Cas Systems/genetics
CpG Islands/genetics
*DNA Demethylation
DNA Methylation
DNA Modification Methylases/genetics/*metabolism
*Epigenesis, Genetic
Fragile X Mental Retardation Protein/genetics
Gene Editing/methods
HEK293 Cells
Humans
Mice
Mixed Function Oxygenases/genetics/*metabolism
NIH 3T3 Cells
Promoter Regions, Genetic/genetics
Proto-Oncogene Proteins/genetics/*metabolism
RNA, Guide/metabolism
Serpins/genetics

@article {pmid34580310,
year = {2021},
author = {Shams, A and Higgins, SA and Fellmann, C and Laughlin, TG and Oakes, BL and Lew, R and Kim, S and Lukarska, M and Arnold, M and Staahl, BT and Doudna, JA and Savage, DF},
title = {Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5664},
pmid = {34580310},
issn = {2041-1723},
support = {K99 GM118909/GM/NIGMS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; R01 GM127463/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; T32 GM066698/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; S10 RR029668/RR/NCRR NIH HHS/United States ; S10 RR027303/RR/NCRR NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism/ultrastructure ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cryoelectron Microscopy ; DNA/metabolism ; Gene Editing/methods ; Humans ; Protein Interaction Domains and Motifs/*genetics ; RNA, Guide/*metabolism ; Single Molecule Imaging ; },
abstract = {Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.},
}

CRISPR-Associated Protein 9/*genetics/metabolism/ultrastructure
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Cryoelectron Microscopy
DNA/metabolism
Gene Editing/methods
Humans
Protein Interaction Domains and Motifs/*genetics
RNA, Guide/*metabolism
Single Molecule Imaging

@article {pmid34669078,
year = {2021},
author = {Yang, FY and Wei, N and Zhang, ZH and Wang, M and Liu, YC and Zhang, LF and Gu, F},
title = {Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD.},
journal = {Biotechnology letters},
volume = {},
number = {},
pages = {},
pmid = {34669078},
issn = {1573-6776},
support = {No.31871247//national natural science foundation of china/ ; },
abstract = {OBJECTIVES: Corynebacterium glutamicum (C. glutamicum) has been harnessed for multi-million-ton scale production of glutamate and lysine. To further increase its amino acid production for fermentation industry, there is an acute need to develop next-generation genome manipulation tool for its metabolic engineering. All reported methods for genome editing triggered with CRISPR-Cas are based on the homologous recombination. While, it requires the generation of DNA repair template, which is a bottle-neck for its extensive application.

RESULTS: In this study, we developed a method for gene knockout in C. glutamicum via CRISPR-Cpf1-coupled non-homologous end-joining (CC-NHEJ). Specifically, CRISPR-Cpf1 introduced double-strand breaks in the genome of C. glutamicum, which was further repaired by ectopically expressed two NHEJ key proteins (Mycobacterium tuberculosis Ku and ligase D). We provide the proof of concept, for CC-NHEJ, by the successful knockout of the crtYf/e gene in C. glutamicum with the efficiency of 22.00 ± 5.56%, or something like that.

CONCLUSION: The present study reported a novel genome manipulation method for C. glutamicum.},
}

@article {pmid34668968,
year = {2021},
author = {Dehdilani, N and Taemeh, SY and Goshayeshi, L and Dehghani, H},
title = {Genetically engineered birds; pre-CRISPR and CRISPR era.},
journal = {Biology of reproduction},
volume = {},
number = {},
pages = {},
doi = {10.1093/biolre/ioab196},
pmid = {34668968},
issn = {1529-7268},
abstract = {Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-CRISPR and CRISPR era in the field of avian genome engineering.},
}

@article {pmid34668031,
year = {2021},
author = {Zhu, T and Zhao, Y},
title = {Correlation between type IIIA CRISPR-Cas system and SCCmec in Staphylococcus epidermidis.},
journal = {Archives of microbiology},
volume = {},
number = {},
pages = {},
pmid = {34668031},
issn = {1432-072X},
support = {81802071//national natural science foundation of china/ ; gxyq2019043//the support program for university outstanding youth talent of anhui province/ ; KJ2020A0602//the key program of educational commission of anhui province/ ; },
abstract = {A subculture of S.epidermidis strain ATCC35984 that is amenable to genetically manipulate was occasionally found in our laboratory. This mutant exhibited susceptibility to methicillin in contrast to its parent strain. To unveil the underlying mechanism, whole-genome sequencing of the mutant was performed. A comparative analysis revealed that a large DNA fragment encompassing the CRISPR-Cas system, type I R-M system and the SCCmec element was deleted from the mutant. The large chromosomal deletion associated with CRISPR-Cas system was also observed to occur spontaneously in S. epidermidis in another independent laboratory, or artificially induced by introducing engineering crRNAs in other bacterial species. These findings imply the CRISPR-Cas systems can affect bacterial genome remodeling through deletion of the integrated MGEs (mobile genetic elements). Further bioinformatics analysis identified a higher carriage rate of SCCmec element in the S. epidermidis strains harboring the CRISPR-Cas system. MLST typing and phylogenetic analysis of those CRIPSR-Cas-positive S. epidermidis strains revealed multiple origins. In addition, distinct types of SCCmec carried in those strains suggested that acquisition of this MGE originated from multiple independent recombination events. Intriguingly, CRISPR-Cas systems are found to be always located in the vicinity of orfX gene among staphylococci. Allelic analysis of CRISPR loci flanking cas genes disclosed that the loci distal to the orfX gene are considerably stable and conserved, which probably serve as recombination hotspot between CRISPR-Cas system and phage or plasmid. Therefore, the findings generally support the notion that incomplete immune protection of CRISPR-Cas system can promote dissemination of its neighboring SCCmec element.},
}

@article {pmid34667116,
year = {2021},
author = {Genzor, P and Konstantinidou, P and Stoyko, D and Manzourolajdad, A and Marlin Andrews, C and Elchert, AR and Stathopoulos, C and Haase, AD},
title = {Cellular abundance shapes function in piRNA-guided genome defense.},
journal = {Genome research},
volume = {},
number = {},
pages = {},
doi = {10.1101/gr.275478.121},
pmid = {34667116},
issn = {1549-5469},
abstract = {Defense against genome invaders universally relies on RNA-guided immunity. Prokaryotic CRISPR-Cas and eukaryotic RNA interference pathways recognize targets by complementary base-pairing, which places the sequences of their guide RNAs at the center of self/nonself discrimination. Here, we explore the sequence space of PIWI-interacting RNAs (piRNAs), the genome defense of animals, and establish functional priority among individual sequences. Our results reveal that only the topmost abundant piRNAs are commonly present in every cell, whereas rare sequences generate cell-to-cell diversity in flies and mice. We identify a skewed distribution of sequence abundance as a hallmark of piRNA populations and show that quantitative differences of more than a 1000-fold are established by conserved mechanisms of biogenesis. Finally, our genomics analyses and direct reporter assays reveal that abundance determines function in piRNA-guided genome defense. Taken together, we identify an effective sequence space and untangle two classes of piRNAs that differ in complexity and function. The first class represents the topmost abundant sequences and drives silencing of genomic parasites. The second class sparsely covers an enormous sequence space. These rare piRNAs cannot function in every cell, every individual, or every generation but create diversity with potential for adaptation in the ongoing arms race with genome invaders.},
}

@article {pmid34666523,
year = {2021},
author = {Weissman, JL and Alseth, EO and Meaden, S and Westra, ER and Fuhrman, JA},
title = {Immune lag is a major cost of prokaryotic adaptive immunity during viral outbreaks.},
journal = {Proceedings. Biological sciences},
volume = {288},
number = {1961},
pages = {20211555},
doi = {10.1098/rspb.2021.1555},
pmid = {34666523},
issn = {1471-2954},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptive immune systems enable bacteria and archaea to efficiently respond to viral pathogens by creating a genomic record of previous encounters. These systems are broadly distributed across prokaryotic taxa, yet are surprisingly absent in a majority of organisms, suggesting that the benefits of adaptive immunity frequently do not outweigh the costs. Here, combining experiments and models, we show that a delayed immune response which allows viruses to transiently redirect cellular resources to reproduction, which we call 'immune lag', is extremely costly during viral outbreaks, even to completely immune hosts. Critically, the costs of lag are only revealed by examining the early, transient dynamics of a host-virus system occurring immediately after viral challenge. Lag is a basic parameter of microbial defence, relevant to all intracellular, post-infection antiviral defence systems, that has to-date been largely ignored by theoretical and experimental treatments of host-phage systems.},
}

@article {pmid34665940,
year = {2021},
author = {Neil, K and Allard, N and Roy, P and Grenier, F and Menendez, A and Burrus, V and Rodrigue, S},
title = {High-efficiency delivery of CRISPR-Cas9 by engineered probiotics enables precise microbiome editing.},
journal = {Molecular systems biology},
volume = {17},
number = {10},
pages = {e10335},
doi = {10.15252/msb.202110335},
pmid = {34665940},
issn = {1744-4292},
support = {159817//Gouvernement du Canada|Canadian Institutes of Health Research (CIHR)/ ; 159817//Fonds de Recherche du Québec - Santé (FRQS)/ ; },
abstract = {Antibiotic resistance threatens our ability to treat infectious diseases, spurring interest in alternative antimicrobial technologies. The use of bacterial conjugation to deliver CRISPR-cas systems programmed to precisely eliminate antibiotic-resistant bacteria represents a promising approach but requires high in situ DNA transfer rates. We have optimized the transfer efficiency of conjugative plasmid TP114 using accelerated laboratory evolution. We hence generated a potent conjugative delivery vehicle for CRISPR-cas9 that can eliminate > 99.9% of targeted antibiotic-resistant Escherichia coli in the mouse gut microbiota using a single dose. We then applied this system to a Citrobacter rodentium infection model, achieving full clearance within four consecutive days of treatment.},
}

@article {pmid34665528,
year = {2021},
author = {Kong, H and Ju, E and Yi, K and Xu, W and Lao, YH and Cheng, D and Zhang, Q and Tao, Y and Li, M and Ding, J},
title = {Advanced Nanotheranostics of CRISPR/Cas for Viral Hepatitis and Hepatocellular Carcinoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2102051},
doi = {10.1002/advs.202102051},
pmid = {34665528},
issn = {2198-3844},
support = {2019YFA0111300//National Key Research and Development Program of China/ ; 2018A050506035//Guangdong Provincial Science and Technology Program/ ; 51903256//National Natural Science Foundation of China/ ; 52022095//National Natural Science Foundation of China/ ; 52073280//National Natural Science Foundation of China/ ; 21907113//National Natural Science Foundation of China/ ; 21875289//National Natural Science Foundation of China/ ; U1501243//National Natural Science Foundation of China/ ; 2019QN01Y131//Guangdong Provincial Pearl River Talents Program/ ; 2016A050503026//Guangdong-Hong Kong Joint Innovation Project/ ; 201704030123//Major Project on the Integration of Industry, Education and Research of Guangzhou City/ ; //Science and Technology Program of Guangzhou/ ; 2019230//Youth Innovation Promotion Association of Chinese Academy of Sciences/ ; },
abstract = {Liver disease, particularly viral hepatitis and hepatocellular carcinoma (HCC), is a global healthcare burden and leads to more than 2 million deaths per year worldwide. Despite some success in diagnosis and vaccine development, there are still unmet needs to improve diagnostics and therapeutics for viral hepatitis and HCC. The emerging clustered regularly interspaced short palindromic repeat/associated proteins (CRISPR/Cas) technology may open up a unique avenue to tackle these two diseases at the genetic level in a precise manner. Especially, liver is a more accessible organ over others from the delivery point of view, and many advanced strategies applied for nanotheranostics can be adapted in CRISPR-mediated diagnostics or liver gene editing. In this review, the focus is on these two aspects of viral hepatitis and HCC applications. An overview on CRISPR editor development and current progress in clinical trials is first given, followed by highlighting the recent advances integrating the merits of gene editing and nanotheranostics. The promising systems that are used in other applications but may hold potentials in liver gene editing are also discussed. This review concludes with the perspectives on rationally designing the next-generation CRISPR approaches and improving the editing performance.},
}

@article {pmid34663942,
year = {2021},
author = {Berríos, KN and Evitt, NH and DeWeerd, RA and Ren, D and Luo, M and Barka, A and Wang, T and Bartman, CR and Lan, Y and Green, AM and Shi, J and Kohli, RM},
title = {Controllable genome editing with split-engineered base editors.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {34663942},
issn = {1552-4469},
support = {F30-HG011578//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01-HG010646//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; T32-GM007170//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01-GM138908//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; K08-CA212299//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
abstract = {DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR-Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.},
}

@article {pmid34663886,
year = {2021},
author = {Lucas-Elío, P and Molina-Quintero, LR and Xu, H and Sánchez-Amat, A},
title = {A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {20564},
pmid = {34663886},
issn = {2045-2322},
support = {BFU2017-85464//Ministerio de Economía, Industria y Competitividad, Gobierno de España/ ; BFU2017-85464//Ministerio de Economía, Industria y Competitividad, Gobierno de España/ ; BFU2017-85464//Ministerio de Economía, Industria y Competitividad, Gobierno de España/ ; 20883/PI/18//Fundación Séneca, Comunidad Autónoma de la Región de Murcia, Spain/ ; 20883/PI/18//Fundación Séneca, Comunidad Autónoma de la Región de Murcia, Spain/ ; 20883/PI/18//Fundación Séneca, Comunidad Autónoma de la Región de Murcia, Spain/ ; R01 GM37949 and R35 GM136216/NH/NIH HHS/United States ; },
abstract = {CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I-F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.},
}

@article {pmid34661427,
year = {2021},
author = {Huang, Y and Zhang, Y and Wu, M and Porter, A and Barrangou, R},
title = {Determination of Factors Driving the Genome Editing Field in the CRISPR Era Using Bibliometrics.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {728-738},
doi = {10.1089/crispr.2021.0001},
pmid = {34661427},
issn = {2573-1602},
abstract = {Over the past two decades, the discovery of CRISPR-Cas immune systems and the repurposing of their effector nucleases as biotechnological tools have revolutionized genome editing. The corresponding work has been captured by 90,000 authors representing 7,600 affiliations in 126 countries, who have published more than 19,000 papers spanning medicine, agriculture, and biotechnology. Here, we use tech mining and an integrated bibliometric and networks framework to investigate the CRISPR literature over three time periods. The analysis identified seminal papers, leading authors, influential journals, and rising applications and topics interconnected through collaborative networks. A core set of foundational topics gave rise to diverging avenues of research and applications, reflecting a bona fide disruptive emerging technology. This analysis illustrates how bibliometrics can identify key factors, decipher rising trends, and untangle emerging applications and technologies that dynamically shape a morphing field, and provides insights into the trajectory of genome editing.},
}

@article {pmid34661426,
year = {2021},
author = {Liu, X and Zhou, X and Li, G and Huang, S and Sun, W and Sun, Q and Li, L and Huang, X and Liu, J and Wang, L},
title = {Editing Properties of Base Editors with SpCas9-NG in Discarded Human Tripronuclear Zygotes.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {710-727},
doi = {10.1089/crispr.2021.0036},
pmid = {34661426},
issn = {2573-1602},
abstract = {DNA base editors, comprising nucleotide deaminases and catalytically impaired Cas9 nickase, have been widely used in various organisms for the efficient creation of point mutations, providing researchers with powerful tools in precise genome editing. However, they have been limited by the scope of the editing. The discovery and engineering of various CRISPR-Cas systems, especially SpCas9 variants xCas9, Cas9-NG, and Cas9-SpRY, have diversified the range of targetable DNA sequences and expanded the targeting scope of genomic base editing. To understand the editing properties comprehensively, we conducted an analysis of the editing properties of adenine base editors and cytosine base editors with xCas9, Cas9-NG, and Cas9-SpRY at endogenous sites with NGN protospacer adjacent motifs (PAM). Then, human genetic disease-associated DNA point mutations were installed at a single site or at dual sites with NGH PAM using base editors with SpCas9-NG (ABEmax-NG and Anc-BE4max-NG [BEs-NG]) in cultured human cell lines. Finally, the editing properties of BEs-NG in discarded human tripronuclear embryos were characterized. This study investigated the editing properties of DNA base editors with a relaxed PAM requirement and demonstrated the potential of BEs-NG in human genetic disease-related research and treatment.},
}

@article {pmid34659160,
year = {2021},
author = {Thomas, P and Abdel-Glil, MY and Eichhorn, I and Semmler, T and Werckenthin, C and Baumbach, C and Murmann, W and Bodenthin-Drauschke, A and Zimmermann, P and Schotte, U and Galante, D and Slavic, D and Wagner, M and Wieler, LH and Neubauer, H and Seyboldt, C},
title = {Genome Sequence Analysis of Clostridium chauvoei Strains of European Origin and Evaluation of Typing Options for Outbreak Investigations.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {732106},
doi = {10.3389/fmicb.2021.732106},
pmid = {34659160},
issn = {1664-302X},
abstract = {Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.},
}

@article {pmid34658640,
year = {2021},
author = {Farhat Ullah, M and Ali, Y and Ramzan Khan, M and Ullah Khan, I and Yan, B and Ijaz Khan, M and Malik, MY},
title = {A review of COVID-19: Treatment Strategies and CRISPR/Cas9 gene editing technology approaches to the coronavirus disease.},
journal = {Saudi journal of biological sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.sjbs.2021.10.020},
pmid = {34658640},
issn = {1319-562X},
abstract = {The new coronavirus SARS-CoV-2 pandemic has put the world on lockdown for the first time in decades. This has wreaked havoc on the global economy, put additional burden on local and global public health resources, and, most importantly, jeopardised human health. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and the CRISPR associated (Cas) protein (CRISPR/Cas) was identified to have structures in E. coli. The most modern of these systems is CRISPR/Cas. Editing the genomes of plants and animals took several years and cost hundreds of thousands of dollars until the CRISPR approach was discovered in 2012. As a result, CRISPR/Cas has piqued the scientific community's attention, particularly for disease diagnosis and treatment, because it is faster, less expensive, and more precise than previous genome editing technologies. Data from gene mutations in specific patients gathered using CRISPR/Cas can aid in the identification of the best treatment strategy for each patient, as well as other research domains such as coronavirus replication in cell culture, such as SARS-CoV2. The implications of the most prevalent driver mutations, on the other hand, are often unknown, making treatment interpretation difficult. For detecting a wide range of target genes, the CRISPR/Cas categories provide highly sensitive and selective tools. Genome-wide association studies are a relatively new strategy to discovering genes involved in human disease when it comes to the next steps in genomic research. Furthermore, CRISPR/Cas provides a method for modifying non-coding portions of the genome, which will help advance whole genome libraries by speeding up the analysis of these poorly defined parts of the genome.},
}

@article {pmid34596650,
year = {2021},
author = {Liu, C and Xu, X and Koivisto, O and Zhou, W and Jacquemet, G and Rosenholm, JM and Zhang, H},
title = {Improving the knock-in efficiency of the MOF-encapsulated CRISPR/Cas9 system through controllable embedding structures.},
journal = {Nanoscale},
volume = {13},
number = {39},
pages = {16525-16532},
doi = {10.1039/d1nr02872c},
pmid = {34596650},
issn = {2040-3372},
mesh = {CRISPR-Cas Systems/genetics ; *Metal-Organic Frameworks ; *Nanostructures ; Plasmids/genetics ; Transfection ; },
abstract = {Appropriate tuning of robust artificial coatings can not only enhance intracellular delivery but also preserve the biological functions of genetic molecules in gene based therapies. Here, we report a strategy to synthesize controllable nanostructures in situ by encapsulating CRISPR/Cas9 plasmids into metal-organic frameworks (MOFs) via biomimetic mineralization. The structure-functionality relationship studies indicate that MOF-coated nanostructures dramatically impact the biological features of the contained plasmids through different embedding structures. The plasmids are homogeneously distributed within the heterogeneous nanoarchitecture and protected from enzymatic degradation. In addition, the plasmid-MOF structure exhibits excellent loading capability, pH-responsive release, and affinity for plasmid binding. Through in vitro assays it was found that the superior MOF vector can greatly enhance cellular endocytosis and endo/lysosomal escape of sheltered plasmids, resulting in successful knock-in of GFP-tagged paxillin genomic sequences in cancer cell lines with high transfection potency compared to our previous studies. Thus, the development of new cost-effective approaches for MOF-based intracellular delivery systems offers an attractive option for overcoming the physiological barriers to CRISPR/Cas9 delivery, which shows great potential for investigating paxillin-associated focal adhesions and signal regulation.},
}

CRISPR-Cas Systems/genetics
*Metal-Organic Frameworks
*Nanostructures
Plasmids/genetics
Transfection

@article {pmid34591643,
year = {2021},
author = {Altae-Tran, H and Kannan, S and Demircioglu, FE and Oshiro, R and Nety, SP and McKay, LJ and Dlakić, M and Inskeep, WP and Makarova, KS and Macrae, RK and Koonin, EV and Zhang, F},
title = {The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases.},
journal = {Science (New York, N.Y.)},
volume = {374},
number = {6563},
pages = {57-65},
doi = {10.1126/science.abj6856},
pmid = {34591643},
issn = {1095-9203},
support = {R01 HG009761/HG/NHGRI NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Conserved Sequence ; DNA Transposable Elements/*genetics ; Endodeoxyribonucleases/*genetics ; *Evolution, Molecular ; Genetic Code ; Genetic Variation ; *RNA, Guide ; RNA, Untranslated/genetics ; },
abstract = {[Figure: see text].},
}

Bacterial Proteins/*genetics
CRISPR-Associated Protein 9/*genetics
CRISPR-Associated Proteins/*genetics
CRISPR-Cas Systems/*genetics
Conserved Sequence
DNA Transposable Elements/*genetics
Endodeoxyribonucleases/*genetics
*Evolution, Molecular
Genetic Code
Genetic Variation
*RNA, Guide
RNA, Untranslated/genetics

@article {pmid34576035,
year = {2021},
author = {Chuang, CK and Lin, WM},
title = {Points of View on the Tools for Genome/Gene Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {18},
pages = {},
pmid = {34576035},
issn = {1422-0067},
support = {MOST 110-2313-B-866-003//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Amino Acid Sequence/genetics ; Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/instrumentation/*trends ; Genome/*genetics ; Humans ; Mutation/genetics ; RNA, Guide/genetics ; },
abstract = {Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.},
}

Amino Acid Sequence/genetics
Animals
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
DNA/genetics
Gene Editing/instrumentation/*trends
Genome/*genetics
Humans
Mutation/genetics
RNA, Guide/genetics

@article {pmid34518536,
year = {2021},
author = {Franke, M and De la Calle-Mustienes, E and Neto, A and Almuedo-Castillo, M and Irastorza-Azcarate, I and Acemel, RD and Tena, JJ and Santos-Pereira, JM and Gómez-Skarmeta, JL},
title = {CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5415},
pmid = {34518536},
issn = {2041-1723},
mesh = {Animals ; Body Patterning/genetics ; CCCTC-Binding Factor/deficiency/*genetics ; CRISPR-Cas Systems ; Chromatin/genetics/metabolism ; Embryo, Nonmammalian/embryology/*metabolism ; Enhancer Elements, Genetic/genetics ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques/*methods ; Organogenesis/genetics ; Promoter Regions, Genetic/genetics ; RNA-Seq/methods ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/deficiency/*genetics ; },
abstract = {Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes.},
}

Animals
Body Patterning/genetics
CCCTC-Binding Factor/deficiency/*genetics
CRISPR-Cas Systems
Chromatin/genetics/metabolism
Embryo, Nonmammalian/embryology/*metabolism
Enhancer Elements, Genetic/genetics
*Gene Expression Regulation, Developmental
Gene Knockout Techniques/*methods
Organogenesis/genetics
Promoter Regions, Genetic/genetics
RNA-Seq/methods
Zebrafish/embryology/*genetics
Zebrafish Proteins/deficiency/*genetics

@article {pmid34513721,
year = {2021},
author = {Lin, H and Li, G and Peng, X and Deng, A and Ye, L and Shi, L and Wang, T and He, J},
title = {The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {590989},
pmid = {34513721},
issn = {2235-2988},
mesh = {Animals ; CRISPR-Cas Systems ; *Communicable Diseases ; Hepatitis B virus/genetics ; Humans ; *Virus Diseases/genetics ; *Viruses/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a set of versatile gene-editing toolkit that perform diverse revolutionary functions in various fields of application such as agricultural practices, food industry, biotechnology, biomedicine, and clinical research. Specially, as a novel antiviral method of choice, CRISPR/Cas9 system has been extensively and effectively exploited to fight against human infectious viruses. Infectious diseases including human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papillomavirus (HPV), and other viruses are still global threats with persistent potential to probably cause pandemics. To facilitate virus removals, the CRISPR/Cas9 system has already been customized to confer new antiviral capabilities into host animals either by modifying host genome or by directly targeting viral inherent factors in the form of DNA. Although several limitations and difficulties still need to be conquered, this technology holds great promises in the treatment of human viral infectious diseases. In this review, we will first present a brief biological feature of CRISPR/Cas9 systems, which includes a description of CRISPR/Cas9 structure and composition; thereafter, we will focus on the investigations and applications that employ CRISPR/Cas9 system to combat several human infectious viruses and discuss challenges and future perspectives of using this new platform in the preclinical and clinical settings as an antiviral strategy.},
}

Animals
CRISPR-Cas Systems
*Communicable Diseases
Hepatitis B virus/genetics
Humans
*Virus Diseases/genetics
*Viruses/genetics

@article {pmid34502338,
year = {2021},
author = {de Vrieze, E and de Bruijn, SE and Reurink, J and Broekman, S and van de Riet, V and Aben, M and Kremer, H and van Wijk, E},
title = {Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502338},
issn = {1422-0067},
support = {PPA-0517-0717-RAD//Foundation Fighting Blindness USA/ ; Project number: 1129//Velux Stiftung/ ; PhD grant//DCMN Radboudumc/ ; //Zeldzame Ziekten Fonds/ ; //Stichting Ushersyndroom/ ; //Stichting Klavertje2/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; *Gene Editing ; Gene Knock-In Techniques/*methods ; Genetic Diseases, Inborn/*genetics ; Genetic Engineering/*methods ; Mutagenesis ; Ribonucleoproteins/genetics/metabolism ; Zebrafish ; Zebrafish Proteins/antagonists & inhibitors/*genetics/metabolism ; },
abstract = {CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3-8% of alleles, and 30-45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.},
}

Animals
*CRISPR-Cas Systems
*Disease Models, Animal
*Gene Editing
Gene Knock-In Techniques/*methods
Genetic Diseases, Inborn/*genetics
Genetic Engineering/*methods
Mutagenesis
Ribonucleoproteins/genetics/metabolism
Zebrafish
Zebrafish Proteins/antagonists & inhibitors/*genetics/metabolism

@article {pmid34502319,
year = {2021},
author = {Hassan, JJ and Lieske, A and Dörpmund, N and Klatt, D and Hoffmann, D and Kleppa, MJ and Kustikova, OS and Stahlhut, M and Schwarzer, A and Schambach, A and Maetzig, T},
title = {A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502319},
issn = {1422-0067},
support = {MA 7010/1//Deutsche Forschungsgemeinschaft/ ; SFB738//Deutsche Forschungsgemeinschaft/ ; MWK: ZN3440//State of Lower Saxony, Germany/ ; REBIRTH EXC62/2//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Proliferation ; *Gene Expression Regulation, Neoplastic ; Group IV Phospholipases A2/*antagonists & inhibitors/genetics ; High-Throughput Screening Assays ; Homeodomain Proteins/genetics/*metabolism ; Humans ; Leukemia, Myeloid, Acute/genetics/metabolism/*pathology ; Myeloid Ecotropic Viral Integration Site 1 Protein/genetics/*metabolism ; Tumor Cells, Cultured ; },
abstract = {HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.},
}

Apoptosis
Biomarkers, Tumor/genetics/*metabolism
*CRISPR-Cas Systems
Cell Proliferation
*Gene Expression Regulation, Neoplastic
Group IV Phospholipases A2/*antagonists & inhibitors/genetics
High-Throughput Screening Assays
Homeodomain Proteins/genetics/*metabolism
Humans
Leukemia, Myeloid, Acute/genetics/metabolism/*pathology
Myeloid Ecotropic Viral Integration Site 1 Protein/genetics/*metabolism
Tumor Cells, Cultured

@article {pmid34502191,
year = {2021},
author = {Lee, HH and Kim, D and Jung, J and Kang, H and Cho, H},
title = {NLRP3 Deficiency in Hepatocellular Carcinoma Enhances Surveillance of NK-92 through a Modulation of MICA/B.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502191},
issn = {1422-0067},
support = {2016R1A6A1A03007648//National Research Foundation of Korea/ ; 2020R1A5A2017323//National Research Foundation of Korea/ ; 2020R1I1A1A01074412//National Research Foundation of Korea/ ; },
mesh = {Animals ; Apoptosis ; Biomarkers, Tumor/genetics/metabolism ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*immunology/metabolism/pathology ; Cell Proliferation ; Cytotoxicity, Immunologic/*immunology ; Disease Models, Animal ; Female ; Gene Expression Regulation, Neoplastic ; Histocompatibility Antigens Class I/genetics/*metabolism ; Humans ; Killer Cells, Natural/*immunology ; Liver Neoplasms/immunology/metabolism/pathology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Monitoring, Immunologic/*methods ; NLR Family, Pyrin Domain-Containing 3 Protein/*deficiency/genetics ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Human hepatocellular carcinoma (HCC) is the most common and even worse at prognosis. The patients with HCC which accompanied by other diseases, such as cirrhosis, can be limited in various treatments, such as chemotherapy, not HCC patients without other diseases. NLRP3 inflammasome plays an important role in the innate immune response, but emerging evidence has indicated that the NLRP3 inflammasome is implicated in all stages of cancer development. Various cells express NLRP3 protein through the autocrine or paracrine signaling in their environment, but NK cells do not. The expanding evidence shows that patients who suffer from liver cancers have a low frequency of natural killer (NK) cells, and the function of these cells is also impaired. Thus, we examined how the expression of NLRP3 in HCC cells affects cancer surveillance by NK cells in a state of a co-culture of both cells. When the expression of NLRP3 in HCC cells was ablated, MICA/B on the surface of HCC cells was upregulated through the lowered expression of matrix metalloproteinase. The expression of MICA on the surface of HCC cells interacted with the NKG2D receptor on NK-92 cells, which led to NK cytotoxicity. Furthermore, in a xenograft mice model, NLRP3 KO HCC cells delayed tumor development and metastasis as well as increased the sensitivity to NK cell cytotoxicity. Taken together, NLRP3 KO in HCC could enhance NK immunosurveillance through an interaction of NKG2D-MICA.},
}

Animals
Apoptosis
Biomarkers, Tumor/genetics/metabolism
CRISPR-Cas Systems
Carcinoma, Hepatocellular/*immunology/metabolism/pathology
Cell Proliferation
Cytotoxicity, Immunologic/*immunology
Disease Models, Animal
Female
Gene Expression Regulation, Neoplastic
Histocompatibility Antigens Class I/genetics/*metabolism
Humans
Killer Cells, Natural/*immunology
Liver Neoplasms/immunology/metabolism/pathology
Mice
Mice, Inbred NOD
Mice, SCID
Monitoring, Immunologic/*methods
NLR Family, Pyrin Domain-Containing 3 Protein/*deficiency/genetics
Tumor Cells, Cultured
Xenograft Model Antitumor Assays

@article {pmid34184636,
year = {2021},
author = {Kulkarni, S and Marquez, J and Date, P and Ventrella, R and Mitchell, BJ and Khokha, MK},
title = {Mechanical stretch scales centriole number to apical area via Piezo1 in multiciliated cells.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34184636},
issn = {2050-084X},
support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; K99 HL133606/HL/NHLBI NIH HHS/United States ; R00 HL133606/HL/NHLBI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; T32 AR060710/AR/NIAMS NIH HHS/United States ; R01 HD102186/HD/NICHD NIH HHS/United States ; R01 GM089970/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Biomechanical Phenomena ; CRISPR-Cas Systems ; Cell Cycle Proteins ; Centrioles/*physiology ; Gene Silencing ; Ion Channels ; Morpholinos ; RNA, Messenger ; Xenopus/embryology ; },
abstract = {How cells count and regulate organelle number is a fundamental question in cell biology. For example, most cells restrict centrioles to two in number and assemble one cilium; however, multiciliated cells (MCCs) synthesize hundreds of centrioles to assemble multiple cilia. Aberration in centriole/cilia number impairs MCC function and can lead to pathological outcomes. Yet how MCCs control centriole number remains unknown. Using Xenopus, we demonstrate that centriole number scales with apical area over a remarkable 40-fold change in size. We find that tensile forces that shape the apical area also trigger centriole amplification based on both cell stretching experiments and disruption of embryonic elongation. Unexpectedly, Piezo1, a mechanosensitive ion channel, localizes near each centriole suggesting a potential role in centriole amplification. Indeed, depletion of Piezo1 affects centriole amplification and disrupts its correlation with the apical area in a tension-dependent manner. Thus, mechanical forces calibrate cilia/centriole number to the MCC apical area via Piezo1. Our results provide new perspectives to study organelle number control essential for optimal cell function.},
}

Animals
Biomechanical Phenomena
CRISPR-Cas Systems
Cell Cycle Proteins
Centrioles/*physiology
Gene Silencing
Ion Channels
Morpholinos
RNA, Messenger
Xenopus/embryology

@article {pmid34143557,
year = {2021},
author = {Zhang, S and Zhang, R and Gao, J and Song, G and Li, J and Li, W and Qi, Y and Li, Y and Li, G},
title = {CRISPR/Cas9-mediated genome editing for wheat grain quality improvement.},
journal = {Plant biotechnology journal},
volume = {19},
number = {9},
pages = {1684-1686},
pmid = {34143557},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Quality Improvement ; RNA, Guide ; *Triticum/genetics ; },
}

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

@article {pmid34114949,
year = {2021},
author = {Li, J and Qin, X and Shi, J and Wang, X and Li, T and Xu, M and Chen, X and Zhao, Y and Han, J and Piao, Y and Zhang, W and Qu, P and Wang, L and Xiang, R and Shi, Y},
title = {A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34114949},
issn = {2050-084X},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Epithelium/immunology ; Female ; Gene Knockdown Techniques ; Humans ; Interleukins/*genetics/metabolism ; Macrophages/immunology ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis/*genetics ; Ovarian Neoplasms/*genetics/immunology/metabolism ; Peritoneal Cavity/pathology ; Receptors, Interleukin/*genetics/metabolism ; Signal Transduction ; Tumor Microenvironment ; },
abstract = {Transcoelomic spread of cancer cells across the peritoneal cavity occurs in most initially diagnosed ovarian cancer (OC) patients and accounts for most cancer-related death. However, how OC cells interact with peritoneal stromal cells to evade the immune surveillance remains largely unexplored. Here, through an in vivo genome-wide CRISPR/Cas9 screen, we identified IL20RA, which decreased dramatically in OC patients during peritoneal metastasis, as a key factor preventing the transcoelomic metastasis of OC. Reconstitution of IL20RA in highly metastatic OC cells greatly suppresses the transcoelomic metastasis. OC cells, when disseminate into the peritoneal cavity, greatly induce peritoneum mesothelial cells to express IL-20 and IL-24, which in turn activate the IL20RA downstream signaling in OC cells to produce mature IL-18, eventually resulting in the polarization of macrophages into the M1-like subtype to clear the cancer cells. Thus, we show an IL-20/IL20RA-mediated crosstalk between OC and mesothelial cells that supports a metastasis-repressing immune microenvironment.},
}

Animals
*CRISPR-Cas Systems
Cell Line, Tumor
Epithelium/immunology
Female
Gene Knockdown Techniques
Humans
Interleukins/*genetics/metabolism
Macrophages/immunology
Mice
Mice, Inbred C57BL
Neoplasm Metastasis/*genetics
Ovarian Neoplasms/*genetics/immunology/metabolism
Peritoneal Cavity/pathology
Receptors, Interleukin/*genetics/metabolism
Signal Transduction
Tumor Microenvironment

@article {pmid34061021,
year = {2021},
author = {Zhao, Q and Kong, Y and Kittredge, A and Li, Y and Shen, Y and Zhang, Y and Tsang, SH and Yang, T},
title = {Distinct expression requirements and rescue strategies for BEST1 loss- and gain-of-function mutations.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34061021},
issn = {2050-084X},
support = {R21 AG050437/AG/NIA NIH HHS/United States ; R01 EY018213/EY/NEI NIH HHS/United States ; U01 EY030580/EY/NEI NIH HHS/United States ; U54 OD020351/OD/NIH HHS/United States ; R01 GM127652/GM/NIGMS NIH HHS/United States ; R01 EY026682/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; P30 EY019007/EY/NEI NIH HHS/United States ; R24 EY027285/EY/NEI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R24 EY028758/EY/NEI NIH HHS/United States ; },
mesh = {Bestrophins/*genetics/metabolism ; CRISPR-Cas Systems ; Chlorides/metabolism ; *Gain of Function Mutation ; Genetic Predisposition to Disease ; Genetic Therapy ; HEK293 Cells ; Humans ; *Loss of Function Mutation ; Membrane Potentials ; Phenotype ; Retinal Degeneration/diagnosis/*genetics/metabolism/therapy ; Retinal Pigment Epithelium/metabolism ; },
abstract = {Genetic mutation of the human BEST1 gene, which encodes a Ca2+-activated Cl- channel (BEST1) predominantly expressed in retinal pigment epithelium (RPE), causes a spectrum of retinal degenerative disorders commonly known as bestrophinopathies. Previously, we showed that BEST1 plays an indispensable role in generating Ca2+-dependent Cl- currents in human RPE cells, and the deficiency of BEST1 function in patient-derived RPE is rescuable by gene augmentation (Li et al., 2017). Here, we report that BEST1 patient-derived loss-of-function and gain-of-function mutations require different mutant to wild-type (WT) molecule ratios for phenotypic manifestation, underlying their distinct epigenetic requirements in bestrophinopathy development, and suggesting that some of the previously classified autosomal dominant mutations actually behave in a dominant-negative manner. Importantly, the strong dominant effect of BEST1 gain-of-function mutations prohibits the restoration of BEST1-dependent Cl- currents in RPE cells by gene augmentation, in contrast to the efficient rescue of loss-of-function mutations via the same approach. Moreover, we demonstrate that gain-of-function mutations are rescuable by a combination of gene augmentation with CRISPR/Cas9-mediated knockdown of endogenous BEST1 expression, providing a universal treatment strategy for all bestrophinopathy patients regardless of their mutation types.},
}

Bestrophins/*genetics/metabolism
CRISPR-Cas Systems
Chlorides/metabolism
*Gain of Function Mutation
Genetic Predisposition to Disease
Genetic Therapy
HEK293 Cells
Humans
*Loss of Function Mutation
Membrane Potentials
Phenotype
Retinal Degeneration/diagnosis/*genetics/metabolism/therapy
Retinal Pigment Epithelium/metabolism

@article {pmid34034859,
year = {2021},
author = {Le Vasseur, M and Friedman, J and Jost, M and Xu, J and Yamada, J and Kampmann, M and Horlbeck, MA and Salemi, MR and Phinney, BS and Weissman, JS and Nunnari, J},
title = {Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34034859},
issn = {2050-084X},
support = {K99 GM130964/GM/NIGMS NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R00 HL133372/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; S10 OD018223/OD/NIH HHS/United States ; R01 GM126081/GM/NIGMS NIH HHS/United States ; R37 GM097432/GM/NIGMS NIH HHS/United States ; K99 HL133372/HL/NHLBI NIH HHS/United States ; },
mesh = {Antimycin A/pharmacology ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electron Transport Complex III/antagonists & inhibitors/genetics/*metabolism ; Endopeptidases/genetics/metabolism ; Genome-Wide Association Study ; Humans ; K562 Cells ; Mitochondria/drug effects/*enzymology/genetics ; Neoplasm Proteins/genetics/*metabolism ; Oxidative Phosphorylation ; Proteolysis ; Repressor Proteins/genetics/*metabolism ; },
abstract = {Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.},
}

Antimycin A/pharmacology
CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Electron Transport Complex III/antagonists & inhibitors/genetics/*metabolism
Endopeptidases/genetics/metabolism
Genome-Wide Association Study
Humans
K562 Cells
Mitochondria/drug effects/*enzymology/genetics
Neoplasm Proteins/genetics/*metabolism
Oxidative Phosphorylation
Proteolysis
Repressor Proteins/genetics/*metabolism

@article {pmid34019632,
year = {2021},
author = {Sun, F and Fang, Y and Zhang, MM and Zhang, RJ and Wu, FY and Yang, RM and Tu, PH and Dong, M and Zhao, SX and Song, HD},
title = {Genetic Manipulation on Zebrafish duox Recapitulate the Clinical Manifestations of Congenital Hypothyroidism.},
journal = {Endocrinology},
volume = {162},
number = {8},
pages = {},
doi = {10.1210/endocr/bqab101},
pmid = {34019632},
issn = {1945-7170},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; *Congenital Hypothyroidism ; *Disease Models, Animal ; Female ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Male ; NADPH Oxidases/*genetics ; Phenotype ; Thyroxine ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {Congenital hypothyroidism (CH) is a highly prevalent but treatable neonatal endocrine disorder. Thyroid dyshormonogenesis is the main cause of congenital hypothyroidism in Chinese CH patients, and DUOX2 is the most frequent mutated gene involved in H2O2 production. In humans, the primary sources for H2O2 production are DUOX1 and DUOX2, while in zebrafish there is only a single orthologue for DUOX1 and DUOX2. In this study, duox mutant zebrafish were generated through knockdown duox by morpholino or knockout duox by CRISPR Cas9. The associated phenotypes were investigated and rescued by thyroxine (T4) treatment. Mutant zebrafish displayed hypothyroid phenotypes including growth retardation, goiter and, infertility. Homozygous mutants in adults also displayed extrathyroidal abnormal phenotypes, including lacking barbels, pigmentation defects, erythema in the opercular region, ragged fins, and delayed scales. All these abnormal phenotypes can be rescued by 10 nM T4 treatment. Strikingly, the fertility of zebrafish was dependent on thyroid hormone; T4 treatment should be continued and cannot be stopped over 2 weeks in hypothyroid zebrafish in order to achieve fertility. Thyroid hormones played a role in the developing and maturing of reproductive cells. Our work indicated that duox mutant zebrafish may provide a model for human congenital hypothyroidism.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems
*Congenital Hypothyroidism
*Disease Models, Animal
Female
Gene Knockdown Techniques
Gene Knockout Techniques
Male
NADPH Oxidases/*genetics
Phenotype
Thyroxine
Zebrafish/*genetics
Zebrafish Proteins/*genetics

@article {pmid33838111,
year = {2021},
author = {Nuñez, JK and Chen, J and Pommier, GC and Cogan, JZ and Replogle, JM and Adriaens, C and Ramadoss, GN and Shi, Q and Hung, KL and Samelson, AJ and Pogson, AN and Kim, JYS and Chung, A and Leonetti, MD and Chang, HY and Kampmann, M and Bernstein, BE and Hovestadt, V and Gilbert, LA and Weissman, JS},
title = {Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2503-2519.e17},
pmid = {33838111},
issn = {1097-4172},
support = {RM1 HG007735/HG/NHGRI NIH HHS/United States ; DP1 CA216873/CA/NCI NIH HHS/United States ; F32 AG063487/AG/NIA NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; R00 GM134154/GM/NIGMS NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; F31 NS115380/NS/NINDS NIH HHS/United States ; K99 GM134154/GM/NIGMS NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Differentiation ; *Cellular Reprogramming ; CpG Islands ; DNA Methylation ; *Epigenesis, Genetic ; *Epigenome ; *Gene Editing ; Gene Silencing ; Histone Code ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Neurons/*cytology/metabolism ; Protein Processing, Post-Translational ; },
abstract = {A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.},
}

*CRISPR-Cas Systems
Cell Differentiation
*Cellular Reprogramming
CpG Islands
DNA Methylation
*Epigenesis, Genetic
*Epigenome
*Gene Editing
Gene Silencing
Histone Code
Humans
Induced Pluripotent Stem Cells/*cytology/metabolism
Neurons/*cytology/metabolism
Protein Processing, Post-Translational

@article {pmid33790381,
year = {2021},
author = {Singh, K and Cornell, CS and Jackson, R and Kabiri, M and Phipps, M and Desai, M and Fogle, R and Ying, X and Anarat-Cappillino, G and Geller, S and Johnson, J and Roberts, E and Malley, K and Devlin, T and DeRiso, M and Berthelette, P and Zhang, YV and Ryan, S and Rao, S and Thurberg, BL and Bangari, DS and Kyostio-Moore, S},
title = {CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7254},
pmid = {33790381},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; *Gene Knockout Techniques ; Male ; Mice ; Mice, Knockout ; Phenylalanine Hydroxylase/*genetics ; Phenylketonurias/*genetics ; },
abstract = {Phenylketonuria (PKU) is an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in buildup of Phe and reduction of Tyr concentration in blood and in the brain, leading to neurological disease and intellectual deficits. Patients exhibit retarded body growth, hypopigmentation, hypocholesterolemia and low levels of neurotransmitters. Here we report first attempt at creating a homozygous Pah knock-out (KO) (Hom) mouse model, which was developed in the C57BL/6 J strain using CRISPR/Cas9 where codon 7 (GAG) in Pah gene was changed to a stop codon TAG. We investigated 2 to 6-month-old, male, Hom mice using comprehensive behavioral and biochemical assays, MRI and histopathology. Age and sex-matched heterozygous Pah-KO (Het) mice were used as control mice, as they exhibit enough PAH enzyme activity to provide Phe and Tyr levels comparable to the wild-type mice. Overall, our findings demonstrate that 6-month-old, male Hom mice completely lack PAH enzyme, exhibit significantly higher blood and brain Phe levels, lower levels of brain Tyr and neurotransmitters along with lower myelin content and have significant behavioral deficit. These mice exhibit phenotypes that closely resemble PKU patients such as retarded body growth, cutaneous hypopigmentation, and hypocholesterolemia when compared to the age- and sex-matched Het mice. Altogether, biochemical, behavioral, and pathologic features of this novel mouse model suggest that it can be used as a reliable translational tool for PKU preclinical research and drug development.},
}

Animals
*CRISPR-Cas Systems
*Disease Models, Animal
*Gene Knockout Techniques
Male
Mice
Mice, Knockout
Phenylalanine Hydroxylase/*genetics
Phenylketonurias/*genetics

@article {pmid33784496,
year = {2021},
author = {Pelletier, JF and Sun, L and Wise, KS and Assad-Garcia, N and Karas, BJ and Deerinck, TJ and Ellisman, MH and Mershin, A and Gershenfeld, N and Chuang, RY and Glass, JI and Strychalski, EA},
title = {Genetic requirements for cell division in a genomically minimal cell.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2430-2440.e16},
doi = {10.1016/j.cell.2021.03.008},
pmid = {33784496},
issn = {1097-4172},
support = {P41 GM103412/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/antagonists & inhibitors/*genetics ; CRISPR-Cas Systems ; Chromosomes, Bacterial/*genetics ; DNA, Bacterial/*genetics ; Genetic Engineering ; *Genome, Bacterial ; Mycoplasma/*genetics ; Synthetic Biology/*methods ; },
abstract = {Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.},
}

Bacterial Proteins/antagonists & inhibitors/*genetics
CRISPR-Cas Systems
Chromosomes, Bacterial/*genetics
DNA, Bacterial/*genetics
Genetic Engineering
*Genome, Bacterial
Mycoplasma/*genetics
Synthetic Biology/*methods

@article {pmid33772993,
year = {2021},
author = {Jiang, Y and An, X and Li, Z and Yan, T and Zhu, T and Xie, K and Liu, S and Hou, Q and Zhao, L and Wu, S and Liu, X and Zhang, S and He, W and Li, F and Li, J and Wan, X},
title = {CRISPR/Cas9-based discovery of maize transcription factors regulating male sterility and their functional conservation in plants.},
journal = {Plant biotechnology journal},
volume = {19},
number = {9},
pages = {1769-1784},
pmid = {33772993},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Plant Breeding ; *Plant Infertility/genetics ; *Transcription Factors/genetics ; *Zea mays/genetics ; },
abstract = {Identifying genic male-sterility (GMS) genes and elucidating their roles are important to unveil plant male reproduction and promote their application in crop breeding. However, compared with Arabidopsis and rice, relatively fewer maize GMS genes have been discovered and little is known about their regulatory pathways underlying anther and pollen development. Here, by sequencing and analysing anther transcriptomes at 11 developmental stages in maize B73, Zheng58 and M6007 inbred lines, 1100 transcription factor (TF) genes were identified to be stably differentially expressed among different developmental stages. Among them, 14 maize TF genes (9 types belonging to five TF families) were selected and performed CRISPR/Cas9-mediated gene mutagenesis, and then, 12 genes in eight types, including ZmbHLH51, ZmbHLH122, ZmTGA9-1/-2/-3, ZmTGA10, ZmMYB84, ZmMYB33-1/-2, ZmPHD11 and ZmLBD10/27, were identified as maize new GMS genes by using DNA sequencing, phenotypic and cytological analyses. Notably, ZmTGA9-1/-2/-3 triple-gene mutants and ZmMYB33-1/-2 double-gene mutants displayed complete male sterility, but their double- or single-gene mutants showed male fertility. Similarly, ZmLBD10/27 double-gene mutant displayed partial male sterility with 32.18% of aborted pollen grains. In addition, ZmbHLH51 was transcriptionally activated by ZmbHLH122 and their proteins were physically interacted. Molecular markers co-segregating with these GMS mutations were developed to facilitate their application in maize breeding. Finally, all 14-type maize GMS TF genes identified here and reported previously were compared on functional conservation and diversification among maize, rice and Arabidopsis. These findings enrich GMS gene and mutant resources for deeply understanding the regulatory network underlying male fertility and for creating male-sterility lines in maize.},
}

CRISPR-Cas Systems
Gene Expression Regulation, Plant
Plant Breeding
*Plant Infertility/genetics
*Transcription Factors/genetics
*Zea mays/genetics

@article {pmid33770501,
year = {2021},
author = {Saito, M and Ladha, A and Strecker, J and Faure, G and Neumann, E and Altae-Tran, H and Macrae, RK and Zhang, F},
title = {Dual modes of CRISPR-associated transposon homing.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2441-2453.e18},
pmid = {33770501},
issn = {1097-4172},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteria/*genetics/metabolism ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements/*physiology ; DNA, Bacterial/genetics/*metabolism ; Gene Editing ; *RNA, Guide ; Recombination, Genetic ; Transposases/genetics/*metabolism ; },
abstract = {Tn7-like transposons have co-opted CRISPR systems, including class 1 type I-F, I-B, and class 2 type V-K. Intriguingly, although these CRISPR-associated transposases (CASTs) undergo robust CRISPR RNA (crRNA)-guided transposition, they are almost never found in sites targeted by the crRNAs encoded by the cognate CRISPR array. To understand this paradox, we investigated CAST V-K and I-B systems and found two distinct modes of transposition: (1) crRNA-guided transposition and (2) CRISPR array-independent homing. We show distinct CAST systems utilize different molecular mechanisms to target their homing site. Type V-K CAST systems use a short, delocalized crRNA for RNA-guided homing, whereas type I-B CAST systems, which contain two distinct target selector proteins, use TniQ for RNA-guided DNA transposition and TnsD for homing to an attachment site. These observations illuminate a key step in the life cycle of CAST systems and highlight the diversity of molecular mechanisms mediating transposon homing.},
}

Bacteria/*genetics/metabolism
Bacterial Proteins/genetics/*metabolism
CRISPR-Associated Proteins/genetics/*metabolism
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Transposable Elements/*physiology
DNA, Bacterial/genetics/*metabolism
Gene Editing
*RNA, Guide
Recombination, Genetic
Transposases/genetics/*metabolism

@article {pmid33762639,
year = {2021},
author = {Wetzel, KS and Guerrero-Bustamante, CA and Dedrick, RM and Ko, CC and Freeman, KG and Aull, HG and Divens, AM and Rock, JM and Zack, KM and Hatfull, GF},
title = {CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {6796},
pmid = {33762639},
issn = {2045-2322},
support = {R35 GM131729/GM/NIGMS NIH HHS/United States ; GT12053/HHMI/Howard Hughes Medical Institute/United States ; R21 AI151264/AI/NIAID NIH HHS/United States ; 54308198/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; Electroporation ; Genetic Engineering/*methods ; RNA, Guide/metabolism ; },
abstract = {Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.},
}

Bacteriophages/*genetics
CRISPR-Cas Systems/*genetics
Electroporation
Genetic Engineering/*methods
RNA, Guide/metabolism

@article {pmid33674748,
year = {2021},
author = {Riedel, M and Berthelsen, MF and Cai, H and Haldrup, J and Borre, M and Paludan, SR and Hager, H and Vendelbo, MH and Wagner, EF and Bakiri, L and Thomsen, MK},
title = {In vivo CRISPR inactivation of Fos promotes prostate cancer progression by altering the associated AP-1 subunit Jun.},
journal = {Oncogene},
volume = {40},
number = {13},
pages = {2437-2447},
pmid = {33674748},
issn = {1476-5594},
support = {741888/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Proliferation ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Mice ; PTEN Phosphohydrolase/*genetics ; Prostate/pathology ; Prostatic Intraepithelial Neoplasia/*genetics/pathology ; Prostatic Neoplasms/*genetics/pathology ; Proto-Oncogene Proteins c-fos/*genetics ; Transcription Factor AP-1/*genetics ; },
abstract = {Prostate cancer is a major global health concern with limited treatment options for advanced disease. Its heterogeneity challenges the identification of crucial driver genes implicated in disease progression. Activating protein-1 (AP-1) transcription factor is associated with cancer since the first identification of its subunits, the proto-oncogenes JUN and FOS. Whereas both JUN and FOS have been implicated in prostate cancer, this study provides the first functional evidence that FOS acts as a tumor suppressor during prostate cancer progression and invasion. Data mining revealed decreased FOS expression in prostate cancer and a further downregulation in metastatic disease, consistent with FOS expression in cell lines derived from different prostate cancer stages. FOS deficiency in prostate cancer cell lines increases cell proliferation and induces oncogenic pathway alterations. Importantly, in vivo CRISPR/Cas9-mediated Fos and Pten double mutation in murine prostate epithelium results in increased proliferation and invasiveness compared to the abrogation of Pten alone. Interestingly, enhanced Jun expression is observed in the murine prostatic intraepithelial neoplasia lacking Fos. CRISPR/Cas9-mediated knockout of Jun combined with Fos and Pten deficiency diminishes the increased proliferation rate in vivo but not the ability to form invasive disease. Overall, we demonstrate that loss of Fos promotes disease progression from clinical latent prostate cancer to advanced disease through accelerated proliferation and invasiveness, partly through Jun.},
}

Animals
CRISPR-Cas Systems
Carcinogenesis/genetics
Cell Proliferation
Disease Models, Animal
Gene Expression Regulation, Neoplastic
Humans
Male
Mice
PTEN Phosphohydrolase/*genetics
Prostate/pathology
Prostatic Intraepithelial Neoplasia/*genetics/pathology
Prostatic Neoplasms/*genetics/pathology
Proto-Oncogene Proteins c-fos/*genetics
Transcription Factor AP-1/*genetics

@article {pmid33667348,
year = {2021},
author = {Hendelman, A and Zebell, S and Rodriguez-Leal, D and Dukler, N and Robitaille, G and Wu, X and Kostyun, J and Tal, L and Wang, P and Bartlett, ME and Eshed, Y and Efroni, I and Lippman, ZB},
title = {Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.},
journal = {Cell},
volume = {184},
number = {7},
pages = {1724-1739.e16},
doi = {10.1016/j.cell.2021.02.001},
pmid = {33667348},
issn = {1097-4172},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Alleles ; Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Chromatin/metabolism ; Gene Expression Regulation, Plant ; *Genes, Plant ; Genetic Pleiotropy/*genetics ; Homeodomain Proteins/*genetics ; Inflorescence/genetics ; Lycopersicon esculentum/genetics ; Mutagenesis ; Plant Development/genetics ; Plant Proteins/*genetics ; Plants, Genetically Modified/genetics/growth & development/metabolism ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid/*genetics ; Solanaceae/genetics/growth & development ; },
abstract = {Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.},
}

Alleles
Arabidopsis/genetics
CRISPR-Cas Systems/genetics
Chromatin/metabolism
Gene Expression Regulation, Plant
*Genes, Plant
Genetic Pleiotropy/*genetics
Homeodomain Proteins/*genetics
Inflorescence/genetics
Lycopersicon esculentum/genetics
Mutagenesis
Plant Development/genetics
Plant Proteins/*genetics
Plants, Genetically Modified/genetics/growth & development/metabolism
Promoter Regions, Genetic
Regulatory Sequences, Nucleic Acid/*genetics
Solanaceae/genetics/growth & development

@article {pmid33448189,
year = {2021},
author = {Feng, Y and Liu, S and Chen, R and Xie, A},
title = {Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing.},
journal = {Journal of Zhejiang University. Science. B},
volume = {22},
number = {1},
pages = {73-86},
pmid = {33448189},
issn = {1862-1783},
mesh = {Base Pairing ; Binding Sites/genetics ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA Repair/genetics/*physiology ; Gene Editing/*methods ; Humans ; Models, Biological ; RNA, Guide/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.},
}

Base Pairing
Binding Sites/genetics
CRISPR-Associated Protein 9/metabolism
*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded
DNA Repair/genetics/*physiology
Gene Editing/*methods
Humans
Models, Biological
RNA, Guide/genetics/metabolism

@article {pmid33375805,
year = {2020},
author = {Jouve de la Barreda, N},
title = {[From transgenesis to gene edition. Bioethical and applied considerations].},
journal = {Cuadernos de bioetica : revista oficial de la Asociacion Espanola de Bioetica y Etica Medica},
volume = {31},
number = {103},
pages = {387-401},
doi = {10.30444/CB.78},
pmid = {33375805},
issn = {1132-1989},
mesh = {Agriculture/methods ; Animal Husbandry/methods ; Animals ; Bacteriological Techniques ; CRISPR-Cas Systems ; Environment ; Food Safety ; Food Security ; Gene Editing/*ethics/legislation & jurisprudence ; Gene Transfer Techniques/*ethics ; Genetic Enhancement/ethics/legislation & jurisprudence ; Global Health ; Humans ; *Organisms, Genetically Modified ; Risk Assessment ; },
abstract = {Transgenesis is a parcel of biotechnology that allows the introduction of genetic information not proper to the genome of living beings, apart from the mechanisms of natural genetic exchange. This made possible to address important applications in bacteria, animals and plants with significant benefits in health, food and environmental aspects. Since its origin, the production of genetically modified organisms (GMOs) caused some controversy due to the possible negative influence of these organisms or their derived products on health and the environment. Over time, genetic modification techniques have renewed, giving way to others of greater precision, simplicity and safety. Currently the CRISPR-Cas9 technique is widely used, which allows to edit, modify or eliminate specific DNA sequences, with multiple applications in the same fields of transgenesis, but adding greater simplicity, security and lower cost. This work presents the main techniques, applications and ethical implications of using these methods and their perspectives in an ever-evolving world. The bacteria for obtaining products of pharmacological interest, new varieties of cultivated plants of higher production, more resistance to growth limiting agents and better nutritional quality and domestic animals modified genetically, offer a set of advantages needed to address the global challenges that affect the lives of many people around the world.},
}

Agriculture/methods
Animal Husbandry/methods
Animals
Bacteriological Techniques
CRISPR-Cas Systems
Environment
Food Safety
Food Security
Gene Editing/*ethics/legislation & jurisprudence
Gene Transfer Techniques/*ethics
Genetic Enhancement/ethics/legislation & jurisprudence
Global Health
Humans
*Organisms, Genetically Modified
Risk Assessment

@article {pmid33375801,
year = {2020},
author = {Santa María D Angelo, R and Quiceno Osorio, JD and Torres Flor, A and Perochena Escalante, AC},
title = {[The crispr / cas9 techniques applied to human genetic enhancement: a biotechnological, anthropological-philosophical and legal dialogue].},
journal = {Cuadernos de bioetica : revista oficial de la Asociacion Espanola de Bioetica y Etica Medica},
volume = {31},
number = {103},
pages = {343-355},
doi = {10.30444/CB.74},
pmid = {33375801},
issn = {1132-1989},
mesh = {Anthropology ; Biotechnology/ethics/legislation & jurisprudence/methods ; *CRISPR-Cas Systems ; Congenital Abnormalities/genetics ; Eugenics/legislation & jurisprudence/methods ; Gene Editing ; Genetic Enhancement/*ethics/legislation & jurisprudence/methods ; Genetic Therapy ; Genome, Human ; Human Characteristics ; Human Rights ; Humans ; *Interdisciplinary Communication ; Internationality ; Mutation ; Philosophy ; Respect ; },
abstract = {The CRISPR editing method is revolutionary. This technique opens the possibility of countless operations in the genome of living beings. However, the risks are high and, in some cases, unpredictable. Therefore, based on an anthropology that recognizes the human person with an inherent dignity that includes the body, this article intends to propose bases for a regulation capable of facing the challenge of CRISPR, especially, given the possibility of confusing its therapeutic resource with the eugenics, also before the imminent risk of unleashing unforeseen consequences such as mutations, malformations and side effects that could be devastating for human life.},
}

Anthropology
Biotechnology/ethics/legislation & jurisprudence/methods
*CRISPR-Cas Systems
Congenital Abnormalities/genetics
Eugenics/legislation & jurisprudence/methods
Gene Editing
Genetic Enhancement/*ethics/legislation & jurisprudence/methods
Genetic Therapy
Genome, Human
Human Characteristics
Human Rights
Humans
*Interdisciplinary Communication
Internationality
Mutation
Philosophy
Respect

@article {pmid33283887,
year = {2021},
author = {Navarro, IC and Tuorto, F and Jordan, D and Legrand, C and Price, J and Braukmann, F and Hendrick, AG and Akay, A and Kotter, A and Helm, M and Lyko, F and Miska, EA},
title = {Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans.},
journal = {The EMBO journal},
volume = {40},
number = {6},
pages = {e105496},
pmid = {33283887},
issn = {1460-2075},
support = {203144/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 11832/CRUK_/Cancer Research UK/United Kingdom ; 104640/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; C6946/A24843/CRUK_/Cancer Research UK/United Kingdom ; C13474/A27826/CRUK_/Cancer Research UK/United Kingdom ; 092096/Z/10/Z/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {5-Methylcytosine/*metabolism ; Adaptation, Physiological/*genetics ; Animals ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/*genetics/physiology ; Cytosine/chemistry ; Gene Editing ; Heat-Shock Response/*genetics ; Hot Temperature ; Leucine/chemistry ; Methyltransferases/metabolism ; Mitochondria/genetics/metabolism ; Proline/chemistry ; Protein Biosynthesis/genetics/physiology ; RNA/chemistry/genetics ; RNA Processing, Post-Transcriptional/*genetics ; Ribosomes/metabolism ; },
abstract = {Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures.},
}

5-Methylcytosine/*metabolism
Adaptation, Physiological/*genetics
Animals
CRISPR-Cas Systems/genetics
Caenorhabditis elegans/*genetics/physiology
Cytosine/chemistry
Gene Editing
Heat-Shock Response/*genetics
Hot Temperature
Leucine/chemistry
Methyltransferases/metabolism
Mitochondria/genetics/metabolism
Proline/chemistry
Protein Biosynthesis/genetics/physiology
RNA/chemistry/genetics
RNA Processing, Post-Transcriptional/*genetics
Ribosomes/metabolism

@article {pmid33258411,
year = {2021},
author = {Hong, Y and Meng, J and He, X and Zhang, Y and Liu, Y and Zhang, C and Qi, H and Luan, Y},
title = {Editing miR482b and miR482c Simultaneously by CRISPR/Cas9 Enhanced Tomato Resistance to Phytophthora infestans.},
journal = {Phytopathology},
volume = {111},
number = {6},
pages = {1008-1016},
doi = {10.1094/PHYTO-08-20-0360-R},
pmid = {33258411},
issn = {0031-949X},
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; *Lycopersicon esculentum/genetics ; *Phytophthora infestans ; Plant Diseases ; },
abstract = {Late blight, caused by Phytophthora infestans, is severely damaging to the global tomato industry. Micro-RNAs (miRNAs) have been widely demonstrated to play vital roles in plant resistance by repressing their target genes. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) method has been continuously improved and extensively applied to edit plant genomes. However, editing multiplex miRNAs by CRISPR/Cas9 in tomato has not been studied yet. We knocked out miR482b and miR482c simultaneously in tomato through the multiplex CRISPR/Cas9 system. Two transgenic plants with silenced miR482b and miR482c simultaneously and one transgenic line with silenced miR482b alone were obtained. Compared with wild-type plants, the disease symptoms of three transgenic plants upon infection were reduced, accompanied by increased expression of their common target nucleotide binding site-leucine-rich repeat genes and decreased levels of reactive oxygen species. Furthermore, silencing miR482b and miR482c simultaneously was more resistant than silencing miR482b alone in tomato. More importantly, we found that knocking out miR482b and miR482c can elicit expression perturbation of other miRNAs, suggesting cross-regulation between miRNAs. Our study demonstrated that editing miR482b and miR482c simultaneously with CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant tomatoes, and cross-regulation between miRNAs may reveal the novel mechanism in tomato-P. infestans interactions.},
}

CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
*Lycopersicon esculentum/genetics
*Phytophthora infestans
Plant Diseases

@article {pmid33238136,
year = {2021},
author = {Sharma, G and Sharma, AR and Bhattacharya, M and Lee, SS and Chakraborty, C},
title = {CRISPR-Cas9: A Preclinical and Clinical Perspective for the Treatment of Human Diseases.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {2},
pages = {571-586},
pmid = {33238136},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Development ; *Gene Editing/methods ; Genetic Diseases, Inborn/*genetics/*therapy ; *Genetic Therapy/methods ; Humans ; },
abstract = {At present, the idea of genome modification has revolutionized the modern therapeutic research era. Genome modification studies have traveled a long way from gene modifications in primary cells to genetic modifications in animals. The targeted genetic modification may result in the modulation (i.e., either upregulation or downregulation) of the predefined gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) is a promising genome-editing tool that has therapeutic potential against incurable genetic disorders by modifying their DNA sequences. In comparison with other genome-editing techniques, CRISPR-Cas9 is simple, efficient, and very specific. This enabled CRISPR-Cas9 genome-editing technology to enter into clinical trials against cancer. Besides therapeutic potential, the CRISPR-Cas9 tool can also be applied to generate genetically inhibited animal models for drug discovery and development. This comprehensive review paper discusses the origin of CRISPR-Cas9 systems and their therapeutic potential against various genetic disorders, including cancer, allergy, immunological disorders, Duchenne muscular dystrophy, cardiovascular disorders, neurological disorders, liver-related disorders, cystic fibrosis, blood-related disorders, eye-related disorders, and viral infection. Finally, we discuss the different challenges, safety concerns, and strategies that can be applied to overcome the obstacles during CRISPR-Cas9-mediated therapeutic approaches.},
}

Animals
CRISPR-Associated Protein 9/genetics/*metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Drug Development
*Gene Editing/methods
Genetic Diseases, Inborn/*genetics/*therapy
*Genetic Therapy/methods
Humans

@article {pmid32761111,
year = {2020},
author = {Shi, X and Tang, T and Lin, Q and Liu, H and Qin, Y and Liang, X and Cong, P and Mo, D and Liu, X and Chen, Y and He, Z},
title = {Efficient generation of bone morphogenetic protein 15-edited Yorkshire pigs using CRISPR/Cas9†.},
journal = {Biology of reproduction},
volume = {103},
number = {5},
pages = {1054-1068},
doi = {10.1093/biolre/ioaa138},
pmid = {32761111},
issn = {1529-7268},
mesh = {Animals ; Bone Morphogenetic Protein 15/*genetics/metabolism ; CRISPR-Cas Systems ; Female ; Fertility/*genetics ; Oocytes/*metabolism ; Ovarian Follicle/*metabolism ; Ovary/*metabolism ; Swine ; },
abstract = {Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.},
}

Animals
Bone Morphogenetic Protein 15/*genetics/metabolism
CRISPR-Cas Systems
Female
Fertility/*genetics
Oocytes/*metabolism
Ovarian Follicle/*metabolism
Ovary/*metabolism
Swine

@article {pmid32541929,
year = {2021},
author = {Lee, EJ and Han, JC and Park, DY and Cho, J and Kee, C},
title = {Effect of connective tissue growth factor gene editing using adeno-associated virus-mediated CRISPR-Cas9 on rabbit glaucoma filtering surgery outcomes.},
journal = {Gene therapy},
volume = {28},
number = {5},
pages = {277-286},
pmid = {32541929},
issn = {1476-5462},
mesh = {Animals ; CRISPR-Cas Systems ; Connective Tissue Growth Factor/genetics ; Dependovirus/genetics ; *Filtering Surgery ; Gene Editing ; *Glaucoma/genetics/surgery ; Rabbits ; },
abstract = {Suppressing excessive wound healing responses is critical to ensure surgical success in glaucoma filtration surgery (GFS). Currently used adjunctive materials can lead to side effects due to the nonselectivity in cell inhibition and may require repeated applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system may become a compelling opportunity in glaucoma surgery due to its high selectivity and permanent effect. Connective tissue growth factor (CTGF) is one of the most potent stimulators of tissue fibrosis in the eye. Therefore, we tested the effect of CTGF suppression using the CRISPR-Cas9 system on GFS fibrosis. We used an adeno-associated virus (AAV)-CRISPR-Cas9 system and confirmed successful CTGF suppression was achieved in fibroblasts in vitro through western blot analysis and deep sequencing. In the in vivo intereye-comparison rabbit GFS model, CRISPR-CTGF-treated eyes showed significantly better survival of the surgery site, less subconjunctival fibrosis, limited collagen deposition, and reduced cellularity than untreated eyes. Our results suggest a new possibility of CRISPR-Cas9-mediated CTGF suppression to improve human GFS outcomes.},
}

Animals
CRISPR-Cas Systems
Connective Tissue Growth Factor/genetics
Dependovirus/genetics
*Filtering Surgery
Gene Editing
*Glaucoma/genetics/surgery
Rabbits

@article {pmid34653714,
year = {2021},
author = {Tian, T and Qiu, Z and Jiang, Y and Zhu, D and Zhou, X},
title = {Exploiting the orthogonal CRISPR-Cas12a/Cas13a trans-cleavage for dual-gene virus detection using a handheld device.},
journal = {Biosensors & bioelectronics},
volume = {196},
number = {},
pages = {113701},
doi = {10.1016/j.bios.2021.113701},
pmid = {34653714},
issn = {1873-4235},
abstract = {Although CRISPR-Cas12a and CRISPR-Cas13a systems work individually effective on gene detection, their multiplex detection capability is limited due to the lack of specific probe cleavage mechanism. Herein we present a high-efficient dual-gene diagnostic technique based on the orthogonal DNA/RNA collateral cleavage mechanism of Cas12a/Cas13a system. In this design, dual-gene amplified products from the multiplex recombinase polymerase amplification (RPA) were simultaneously detected by Cas12a and Cas13a assay in a single tube. The resulting orthogonal DNA/RNA collateral cleavage can specifically illuminate two spectral differentiated DNA and RNA probes, respectively. By integrating with the smartphone-based fluorescence readout, a portable detection platform is achieved. As a proof-of-concept, reliable dual-gene detection of SARS-CoV-2 and African Swine fever virus (ASFV) were demonstrated, exhibiting 100% sensitivity and specificity for clinical samples analysis (32 swab specimens for SARS-CoV-2 and 35 ASFV suspected swine blood samples). This developed portable dual-gene detection platform can provide accurate point-of-care screening of infectious diseases in resources-limited settings.},
}

@article {pmid34653201,
year = {2021},
author = {Mall, EM and Lecanda, A and Drexler, HCA and Raz, E and Schöler, HR and Schlatt, S},
title = {Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs.},
journal = {PloS one},
volume = {16},
number = {10},
pages = {e0258427},
doi = {10.1371/journal.pone.0258427},
pmid = {34653201},
issn = {1932-6203},
abstract = {The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3, PRDM1) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.},
}

@article {pmid34652874,
year = {2021},
author = {Morishita, M and Arimoto-Matsuzaki, K and Kitamura, M and Niimura, K and Iwasa, H and Maruyama, J and Hiraoka, Y and Yamamoto, K and Kitagawa, M and Miyamura, N and Nishina, H and Hata, Y},
title = {Characterization of mouse embryonic fibroblasts derived from Rassf6 knockout mice reveals the implication of Rassf6 in the regulation of NF-κB signaling.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {},
number = {},
pages = {},
doi = {10.1111/gtc.12901},
pmid = {34652874},
issn = {1365-2443},
abstract = {RASSF6 is a member of the tumor suppressor Ras-association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor-suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis revealed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-Dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.},
}

@article {pmid34570096,
year = {2021},
author = {Kojin, BB and Tsujimoto, H and Jakes, E and O'Leary, S and Adelman, ZN},
title = {Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {175},
pages = {},
doi = {10.3791/63008},
pmid = {34570096},
issn = {1940-087X},
mesh = {*Aedes/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; Humans ; Mutagenesis ; },
abstract = {Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.},
}

*Aedes/genetics
Animals
*CRISPR-Cas Systems/genetics
Gene Editing
Genome
Humans
Mutagenesis

@article {pmid34542533,
year = {2021},
author = {Šafarič Tepeš, P and Sordella, R},
title = {A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {175},
pages = {},
doi = {10.3791/60685},
pmid = {34542533},
issn = {1940-087X},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Endonucleases ; Gene Editing ; Genome ; Mutagenesis, Site-Directed ; },
abstract = {The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.},
}

*CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems/genetics
Endonucleases
Gene Editing
Genome
Mutagenesis, Site-Directed

@article {pmid34338666,
year = {2021},
author = {He, X and Ma, Q and Jian, B and Liu, Y and Wu, Q and Chen, M and Feng, Q and Zhao, P and Liu, L},
title = {Microsurgical Obstruction of Testes Fusion in Spodoptera litura.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {173},
pages = {},
doi = {10.3791/62524},
pmid = {34338666},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Endonucleases ; Gene Editing ; Male ; Spermatozoa ; Spodoptera/genetics ; *Testis/surgery ; },
abstract = {Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9, a physical barrier was microsurgically inserted between the testes of Spodoptera litura to study the impact of this microsurgery on its growth and reproduction. After inserting aluminum foil between the testes, insect molting during metamorphosis proceeded normally. Insect growth and development were not remarkably altered; however, the number of sperm bundles changed if testes fusion was stopped by the microsurgery. These findings imply that blocking testicular fusion can influence male reproduction capability. The method can be further applied to interrupt communication between organs to study the function of specific signaling pathways. Compared to conventional surgery, microsurgery only requires freezing anesthetization, which is preferable to carbon dioxide anesthetization. Microsurgery also minimizes the surgery site area and facilitates wound healing. However, the selection of materials with specific functions needs further investigation. Avoiding tissue injury is crucial when making incisions during the operation.},
}

Animals
*CRISPR-Cas Systems
Endonucleases
Gene Editing
Male
Spermatozoa
Spodoptera/genetics
*Testis/surgery

@article {pmid34279489,
year = {2021},
author = {Ai, D and Wang, B and Fan, Z and Fu, Y and Yu, C and Wang, G},
title = {Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner).},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {173},
pages = {},
doi = {10.3791/62068},
pmid = {34279489},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Microinjections ; *Moths/genetics ; RNA, Guide/genetics ; },
abstract = {The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.},
}

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

@article {pmid34251367,
year = {2021},
author = {Carballar-Lejarazú, R and Pham, TB and Kelsey, A and Tushar, T and James, AA},
title = {Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {172},
pages = {},
doi = {10.3791/62607},
pmid = {34251367},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Culicidae/genetics ; *Gene Editing ; INDEL Mutation ; Polymerase Chain Reaction ; RNA, Guide ; },
abstract = {Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.},
}

Animals
CRISPR-Cas Systems/genetics
*Culicidae/genetics
*Gene Editing
INDEL Mutation
Polymerase Chain Reaction
RNA, Guide

@article {pmid34152311,
year = {2021},
author = {Graham, S and Gao, L and Wang, R},
title = {Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {172},
pages = {},
doi = {10.3791/61618},
pmid = {34152311},
issn = {1940-087X},
mesh = {Animals ; Antibodies ; CRISPR-Cas Systems ; *Colitis/chemically induced/genetics ; Endonucleases ; *Gene Editing ; Mice ; },
abstract = {The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.},
}

Animals
Antibodies
CRISPR-Cas Systems
*Colitis/chemically induced/genetics
Endonucleases
*Gene Editing
Mice

@article {pmid34049503,
year = {2021},
author = {Ramaker, RC and Hardigan, AA and Gordon, ER and Wright, CA and Myers, RM and Cooper, SJ},
title = {Pooled CRISPR screening in pancreatic cancer cells implicates co-repressor complexes as a cause of multiple drug resistance via regulation of epithelial-to-mesenchymal transition.},
journal = {BMC cancer},
volume = {21},
number = {1},
pages = {632},
pmid = {34049503},
issn = {1471-2407},
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Carcinoma, Pancreatic Ductal/*drug therapy/genetics/mortality/pathology ; Cell Line, Tumor ; Cell Movement/genetics ; Chromatin Immunoprecipitation Sequencing ; Co-Repressor Proteins/genetics/metabolism ; Drug Resistance, Multiple/*genetics ; Drug Resistance, Neoplasm/*genetics ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Histone Deacetylase 1/genetics/metabolism ; Humans ; Pancreatic Neoplasms/*drug therapy/genetics/mortality/pathology ; RNA-Seq ; },
abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) patients suffer poor outcomes, including a five-year survival of below 10%. Poor outcomes result in part from therapeutic resistance that limits the impact of cytotoxic first-line therapy. Novel therapeutic approaches are needed, but currently no targeted therapies exist to treat PDAC.

METHODS: To assess cellular resistance mechanisms common to four cytotoxic chemotherapies (gemcitabine, 5-fluorouracil, irinotecan, and oxaliplatin) used to treat PDAC patients, we performed four genome-wide CRISPR activation (CRISPRact) and CRISPR knock-out (CRISPRko) screens in two common PDAC cell lines (Panc-1 and BxPC3). We used pathway analysis to identify gene sets enriched among our hits and conducted RNA-sequencing and chromatin immunoprecipitation-sequencing (ChIP-seq) to characterize top hits from our screen. We used scratch assays to assess changes in cellular migration with HDAC1 overexpression.

RESULTS: Our data revealed activation of ABCG2, a well-described efflux pump, as the most consistent mediator of resistance in each of our screens. CRISPR-mediated activation of genes involved in transcriptional co-repressor complexes also conferred resistance to multiple drugs. Expression of many of these genes, including HDAC1, is associated with reduced survival in PDAC patients. Up-regulation of HDAC1 in vitro increased promoter occupancy and expression of several genes involved in the epithelial-to-mesenchymal transition (EMT). These cells also displayed phenotypic changes in cellular migration consistent with activation of the EMT pathway. The expression changes resulting from HDAC1 activation were also observed with activation of several other co-repressor complex members. Finally, we developed a publicly available analysis tool, PancDS, which integrates gene expression profiles with our screen results to predict drug sensitivity in resected PDAC tumors and cell lines.

CONCLUSION: Our results provide a comprehensive resource for identifying cellular mechanisms of drug resistance in PDAC, mechanistically implicate HDAC1, and co-repressor complex members broadly, in multi-drug resistance, and provide an analytical tool for predicting treatment response in PDAC tumors and cell lines.},
}

Antineoplastic Agents/*pharmacology/therapeutic use
CRISPR-Cas Systems/genetics
Carcinoma, Pancreatic Ductal/*drug therapy/genetics/mortality/pathology
Cell Line, Tumor
Cell Movement/genetics
Chromatin Immunoprecipitation Sequencing
Co-Repressor Proteins/genetics/metabolism
Drug Resistance, Multiple/*genetics
Drug Resistance, Neoplasm/*genetics
Epithelial-Mesenchymal Transition/genetics
Gene Expression Regulation, Neoplastic
Gene Knockdown Techniques
Histone Deacetylase 1/genetics/metabolism
Humans
Pancreatic Neoplasms/*drug therapy/genetics/mortality/pathology
RNA-Seq

@article {pmid33982289,
year = {2021},
author = {Velíšková, J and Marra, C and Liu, Y and Shekhar, A and Park, DS and Iatckova, V and Xie, Y and Fishman, GI and Velíšek, L and Goldfarb, M},
title = {Early onset epilepsy and sudden unexpected death in epilepsy with cardiac arrhythmia in mice carrying the early infantile epileptic encephalopathy 47 gain-of-function FHF1(FGF12) missense mutation.},
journal = {Epilepsia},
volume = {62},
number = {7},
pages = {1546-1558},
pmid = {33982289},
issn = {1528-1167},
support = {R01 NS092786/NS/NINDS NIH HHS/United States ; R01 HL142498/HL/NHLBI NIH HHS/United States ; R01 HL105983/HL/NHLBI NIH HHS/United States ; P30 CA196521/CA/NCI NIH HHS/United States ; F31 HL132438/HL/NHLBI NIH HHS/United States ; },
mesh = {Age of Onset ; Animals ; Animals, Newborn ; Arrhythmias, Cardiac/etiology/*genetics ; CRISPR-Cas Systems ; Electrocardiography ; Electroencephalography ; Epilepsy, Tonic-Clonic/genetics ; Fibroblast Growth Factors/*genetics ; Genotype ; Humans ; Mice ; Mice, Transgenic ; Mutation, Missense/genetics ; Oligonucleotides ; Seizures/etiology/genetics ; Spasms, Infantile/*genetics ; *Sudden Unexpected Death in Epilepsy ; Voltage-Gated Sodium Channels/metabolism ; },
abstract = {OBJECTIVE: Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice.

METHODS: The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1R52H /F+ mice. Spontaneous epileptiform events in Fhf1R52H /+ mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure-induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1BR52H protein was assayed by voltage-clamp recordings of FHF-deficient mouse cardiomyocytes infected with adenoviruses expressing wild-type FHF1B or FHF1BR52H protein.

RESULTS: All Fhf1R52H /+ mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26 days of age. EEG recordings in 19-20-day-old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2-53 s after lethal seizure onset, heart rate abruptly declined from 572 ± 16 bpm to 108 ± 15 bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1BR52H in cardiomyocytes induced a 15-mV depolarizing shift in voltage of steady-state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1R52H /+ mice prior to seizure.

SIGNIFICANCE: The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Nav 1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Nav 1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.},
}

Age of Onset
Animals
Animals, Newborn
Arrhythmias, Cardiac/etiology/*genetics
CRISPR-Cas Systems
Electrocardiography
Electroencephalography
Epilepsy, Tonic-Clonic/genetics
Fibroblast Growth Factors/*genetics
Genotype
Humans
Mice
Mice, Transgenic
Mutation, Missense/genetics
Oligonucleotides
Seizures/etiology/genetics
Spasms, Infantile/*genetics
*Sudden Unexpected Death in Epilepsy
Voltage-Gated Sodium Channels/metabolism

@article {pmid33964614,
year = {2021},
author = {Slaymaker, IM and Gaudelli, NM},
title = {Engineering Cas9 for human genome editing.},
journal = {Current opinion in structural biology},
volume = {69},
number = {},
pages = {86-98},
doi = {10.1016/j.sbi.2021.03.004},
pmid = {33964614},
issn = {1879-033X},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; },
abstract = {Since the initial reports describing CRISPR-Cas9, labs across the globe have leveraged this valuable gene editing tool to alter the genomes of living cells. With the goal of generating more precise and efficient genome changes, scientists and engineers have mutated, evolved, and covalently altered Cas9 in order to predictably edit the genetic code. Here, we highlight recent advancements and contributions to the growing field of Cas9 engineering. We present key aspects of Cas9 engineering efforts focused on sgRNA manipulation, PAM-recognition, specificity, deaminase fusions, reverse-transcriptase fusions, and structural rearrangements of this important gene-modifying tool.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing
Humans

@article {pmid33754479,
year = {2021},
author = {Miao, Y and Xia, Y and Kong, Y and Zhu, H and Mei, H and Li, P and Feng, H and Xun, W and Xu, Z and Zhang, N and Liu, D and Shen, Q and Zhang, R},
title = {Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9-based cyclical multiple genes coediting in filamentous fungi.},
journal = {Environmental microbiology},
volume = {23},
number = {6},
pages = {2937-2954},
doi = {10.1111/1462-2920.15477},
pmid = {33754479},
issn = {1462-2920},
mesh = {*CRISPR-Cas Systems/genetics ; Fungi/genetics ; Gene Editing ; Homologous Recombination ; *RNA, Guide/genetics ; },
abstract = {Deciphering the complex cellular behaviours and advancing the biotechnology applications of filamentous fungi increase the requirement for genetically manipulating a large number of target genes. The current strategies cannot cyclically coedit multiple genes simultaneously. In this study, we firstly revealed the existence of diverse homologous recombination (HR) types in marker-free editing of filamentous fungi, and then, demonstrated that sgRNA efficiency-mediated competitive inhibition resulted in the low integration of multiple genetic sites during coediting, which are the two major obstacles to limit the efficiency of cyclically coediting of multiple genes. To overcome these obstacles, we developed a biased cutting strategy by Cas9 to greatly enhance the desired HR type and applied a new selection marker labelling strategy for multiple donor DNAs, in which only the donor DNA with the lowest sgRNA efficiency was labelled. Combined with these strategies, we successfully developed a convenient method for cyclically coediting multiple genes in different filamentous fungi. In addition, diverse HRs resulted in a useful and convenient one-step approach for gene functional study combining both gene disruption and complementation. This research provided both a useful one-step approach for gene functional study and an efficient strategy for cyclically coediting multiple genes in filamentous fungi.},
}

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

@article {pmid33724663,
year = {2021},
author = {Ah-Fong, AMV and Boyd, AM and Matson, MEH and Judelson, HS},
title = {A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor.},
journal = {Molecular plant pathology},
volume = {22},
number = {6},
pages = {737-752},
pmid = {33724663},
issn = {1364-3703},
mesh = {Alleles ; CRISPR-Cas Systems ; Chromatin/genetics ; *Gene Editing ; Genomics ; Phytophthora infestans/*genetics/physiology ; Plant Diseases/*parasitology ; Solanum tuberosum/*parasitology ; },
abstract = {Phytophthora infestans is a destructive pathogen of potato and a model for investigations of oomycete biology. The successful application of a CRISPR gene editing system to P. infestans is so far unreported. We discovered that it is difficult to express CRISPR/Cas9 but not a catalytically inactive form in transformants, suggesting that the active nuclease is toxic. We were able to achieve editing with CRISPR/Cas12a using vectors in which the nuclease and its guide RNA were expressed from a single transcript. Using the elicitor gene Inf1 as a target, we observed editing of one or both alleles in up to 13% of transformants. Editing was more efficient when guide RNA processing relied on the Cas12a direct repeat instead of ribozyme sequences. INF1 protein was not made when both alleles were edited in the same transformant, but surprisingly also when only one allele was altered. We discovered that the isolate used for editing, 1306, exhibited monoallelic expression of Inf1 due to insertion of a copia-like element in the promoter of one allele. The element exhibits features of active retrotransposons, including a target site duplication, long terminal repeats, and an intact polyprotein reading frame. Editing occurred more often on the transcribed allele, presumably due to differences in chromatin structure. The Cas12a system not only provides a tool for modifying genes in P. infestans, but also for other members of the genus by expanding the number of editable sites. Our work also highlights a natural mechanism that remodels oomycete genomes.},
}

Alleles
CRISPR-Cas Systems
Chromatin/genetics
*Gene Editing
Genomics
Phytophthora infestans/*genetics/physiology
Plant Diseases/*parasitology
Solanum tuberosum/*parasitology

@article {pmid33669346,
year = {2021},
author = {Brakebusch, C},
title = {CRISPR Genome Editing: How to Make a Fantastic Method Even Better.},
journal = {Cells},
volume = {10},
number = {2},
pages = {},
pmid = {33669346},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing/methods ; Humans ; },
abstract = {CRISPR genome editing describes targeted mutagenesis involving a programmable DNA scissor consisting of a protein (Cas9) bound to a short RNA [...].},
}

Animals
CRISPR-Cas Systems/*genetics
*Gene Editing/methods
Humans

@article {pmid33459978,
year = {2021},
author = {Cao, Y and Zhou, H and Zhou, X and Li, F},
title = {Conferring Resistance to Plant RNA Viruses with the CRISPR/CasRx System.},
journal = {Virologica Sinica},
volume = {36},
number = {4},
pages = {814-817},
pmid = {33459978},
issn = {1995-820X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Diseases ; *Plant Viruses/genetics ; *RNA Viruses/genetics ; RNA, Plant ; },
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Plant Diseases
*Plant Viruses/genetics
*RNA Viruses/genetics
RNA, Plant

@article {pmid32978063,
year = {2020},
author = {Baeten, P and Hellings, N and Broux, B},
title = {In Vitro Tailoring of Regulatory T Cells Prior to Cell Therapy.},
journal = {Trends in molecular medicine},
volume = {26},
number = {11},
pages = {1059-1060},
doi = {10.1016/j.molmed.2020.08.008},
pmid = {32978063},
issn = {1471-499X},
mesh = {Autoimmune Diseases/immunology/therapy ; CRISPR-Cas Systems ; *Cell- and Tissue-Based Therapy/methods ; Gene Editing ; Humans ; Immunotherapy, Adoptive ; Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Receptors, Chimeric Antigen ; T-Lymphocytes, Regulatory/*immunology/*metabolism ; },
}

Autoimmune Diseases/immunology/therapy
CRISPR-Cas Systems
*Cell- and Tissue-Based Therapy/methods
Gene Editing
Humans
Immunotherapy, Adoptive
Receptors, Antigen, T-Cell/genetics/immunology/metabolism
Receptors, Chimeric Antigen
T-Lymphocytes, Regulatory/*immunology/*metabolism

@article {pmid34644300,
year = {2021},
author = {Forsberg, KJ and Schmidtke, DT and Werther, R and Uribe, RV and Hausman, D and Sommer, MOA and Stoddard, BL and Kaiser, BK and Malik, HS},
title = {The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity.},
journal = {PLoS biology},
volume = {19},
number = {10},
pages = {e3001428},
doi = {10.1371/journal.pbio.3001428},
pmid = {34644300},
issn = {1545-7885},
abstract = {To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid-binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs.},
}

@article {pmid34643870,
year = {2021},
author = {Bhagwat, AC and Patil, AM and Saroj, SD},
title = {CRISPR/Cas 9-Based Editing in the Production of Bioactive Molecules.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {34643870},
issn = {1559-0305},
support = {598515-EPP-1-2018-1-IN-EPPKA2-CBHE-JP//erasmus+/ ; },
abstract = {Plants, fungi, and bacteria synthesize a wide range of secondary metabolites that exhibit diverse biological activities. These bioactives, due to their potential benefits in research and therapeutics, have gained immense industrial importance. There is a need to synthesize these bioactives at significantly higher concentrations using cost-effective measures to be economically viable. However, the broader study of industrially important secondary metabolites has been hindered, thus, far due to a shortage of reliable, comparatively easy, and highly effective gene manipulation techniques. With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas), there is a revolution in the field of genetic engineering. CRISPR/Cas system, due to its simplicity and ease of use. This has widened its application in plant breeding, strain improvement, and engineering the metabolic pathways involved in the biochemical synthesis of industrially valuable bioactive. This review briefly introduces the CRISPR/Cas9 system and summarizes the applications of CRISPR/Cas9-mediated editing tools for the production of plant and fungal-derived bioactives.},
}

@article {pmid34643819,
year = {2021},
author = {Gao, Y and Gao, K and Yang, H},
title = {Correction to: CRISPR/Cas: a potential gene-editing tool in the nervous system.},
journal = {Cell regeneration (London, England)},
volume = {10},
number = {1},
pages = {35},
doi = {10.1186/s13619-021-00095-3},
pmid = {34643819},
issn = {2045-9769},
}

@article {pmid34639221,
year = {2021},
author = {Kaur, M and Manchanda, P and Kalia, A and Ahmed, FK and Nepovimova, E and Kuca, K and Abd-Elsalam, KA},
title = {Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
doi = {10.3390/ijms221910882},
pmid = {34639221},
issn = {1422-0067},
support = {SR/WOS-A/LS-610/2016//Department of Science and Technology/ ; VT2019-2021//University of Hradec Kralove/ ; UHHK, 00179906//MH CZ-DRO/ ; },
abstract = {Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.},
}

@article {pmid34638696,
year = {2021},
author = {González Castro, N and Bjelic, J and Malhotra, G and Huang, C and Alsaffar, SH},
title = {Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
doi = {10.3390/ijms221910355},
pmid = {34638696},
issn = {1422-0067},
abstract = {Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.},
}

@article {pmid34635250,
year = {2022},
author = {Zhang, K and Fan, Z and Huang, Y and Ding, Y and Xie, M},
title = {A strategy combining 3D-DNA Walker and CRISPR-Cas12a trans-cleavage activity applied to MXene based electrochemiluminescent sensor for SARS-CoV-2 RdRp gene detection.},
journal = {Talanta},
volume = {236},
number = {},
pages = {122868},
doi = {10.1016/j.talanta.2021.122868},
pmid = {34635250},
issn = {1873-3573},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; DNA ; Gold ; Humans ; *Metal Nanoparticles ; RNA, Viral ; RNA-Dependent RNA Polymerase ; SARS-CoV-2 ; },
abstract = {Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.},
}

*COVID-19
CRISPR-Cas Systems/genetics
DNA
Gold
Humans
*Metal Nanoparticles
RNA, Viral
RNA-Dependent RNA Polymerase
SARS-CoV-2

@article {pmid34542296,
year = {2021},
author = {Nouri, R and Jiang, Y and Tang, Z and Lian, XL and Guan, W},
title = {Detection of SARS-CoV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores.},
journal = {Nano letters},
volume = {21},
number = {19},
pages = {8393-8400},
doi = {10.1021/acs.nanolett.1c02974},
pmid = {34542296},
issn = {1530-6992},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; *Nanopores ; Nucleic Acid Amplification Techniques ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/μL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.},
}

*COVID-19
CRISPR-Cas Systems/genetics
Humans
*Nanopores
Nucleic Acid Amplification Techniques
SARS-CoV-2
Sensitivity and Specificity

@article {pmid34374341,
year = {2021},
author = {Weißenbruch, K and Grewe, J and Hippler, M and Fladung, M and Tremmel, M and Stricker, K and Schwarz, US and Bastmeyer, M},
title = {Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34374341},
issn = {2050-084X},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/physiology ; *Elasticity ; Homeostasis ; Humans ; Models, Theoretical ; Myosin Type II/classification/genetics/*metabolism ; Nonmuscle Myosin Type IIA/genetics/*metabolism ; Nonmuscle Myosin Type IIB/genetics/*metabolism ; Protein Isoforms ; },
abstract = {Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement/physiology
*Elasticity
Homeostasis
Humans
Models, Theoretical
Myosin Type II/classification/genetics/*metabolism
Nonmuscle Myosin Type IIA/genetics/*metabolism
Nonmuscle Myosin Type IIB/genetics/*metabolism
Protein Isoforms

@article {pmid34181658,
year = {2021},
author = {Xue, WH and Xu, N and Chen, SJ and Liu, XY and Zhang, JL and Xu, HJ},
title = {Neofunctionalization of a second insulin receptor gene in the wing-dimorphic planthopper, Nilaparvata lugens.},
journal = {PLoS genetics},
volume = {17},
number = {6},
pages = {e1009653},
pmid = {34181658},
issn = {1553-7404},
mesh = {Adaptation, Physiological/genetics ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Energy Metabolism/genetics ; *Evolution, Molecular ; Gene Dosage ; Gene Editing/methods ; *Gene Expression Regulation, Developmental ; Hemiptera/anatomy & histology/*genetics/growth & development/metabolism ; Insect Proteins/*genetics/metabolism ; Longevity/genetics ; Nymph/genetics/growth & development/metabolism ; Phenotype ; Receptor, Insulin/*genetics/metabolism ; Signal Transduction ; Starvation/genetics/metabolism ; Wings, Animal/anatomy & histology/growth & development/*metabolism ; },
abstract = {A single insulin receptor (InR) gene has been identified and extensively studied in model species ranging from nematodes to mice. However, most insects possess additional copies of InR, yet the functional significance, if any, of alternate InRs is unknown. Here, we used the wing-dimorphic brown planthopper (BPH) as a model system to query the role of a second InR copy in insects. NlInR2 resembled the BPH InR homologue (NlInR1) in terms of nymph development and reproduction, but revealed distinct regulatory roles in fuel metabolism, lifespan, and starvation tolerance. Unlike a lethal phenotype derived from NlInR1 null, homozygous NlInR2 null mutants were viable and accelerated DNA replication and cell proliferation in wing cells, thus redirecting short-winged-destined BPHs to develop into long-winged morphs. Additionally, the proper expression of NlInR2 was needed to maintain symmetric vein patterning in wings. Our findings provide the first direct evidence for the regulatory complexity of the two InR paralogues in insects, implying the functionally independent evolution of multiple InRs in invertebrates.},
}

Adaptation, Physiological/genetics
Animals
Base Sequence
CRISPR-Cas Systems
Energy Metabolism/genetics
*Evolution, Molecular
Gene Dosage
Gene Editing/methods
*Gene Expression Regulation, Developmental
Hemiptera/anatomy & histology/*genetics/growth & development/metabolism
Insect Proteins/*genetics/metabolism
Longevity/genetics
Nymph/genetics/growth & development/metabolism
Phenotype
Receptor, Insulin/*genetics/metabolism
Signal Transduction
Starvation/genetics/metabolism
Wings, Animal/anatomy & histology/growth & development/*metabolism

@article {pmid34122418,
year = {2021},
author = {Dudek, AM and Porteus, MH},
title = {Answered and Unanswered Questions in Early-Stage Viral Vector Transduction Biology and Innate Primary Cell Toxicity for Ex-Vivo Gene Editing.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {660302},
pmid = {34122418},
issn = {1664-3224},
support = {F32 HL154667/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Capsid/metabolism ; Capsid Proteins/genetics ; *DNA Damage ; Dependovirus/*genetics/immunology ; *Gene Editing ; Genetic Therapy ; *Genetic Vectors ; Genome, Viral ; Humans ; *Immunity, Innate ; Mice ; },
abstract = {Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair. Many groups have used this strategy in development of therapies for blood and immune disorders such as sickle-cell anemia and severe-combined immunodeficiency. However, recent events have called into question the immunogenicity of AAV as a gene therapy vector and the safety profile dictated by the immune response to this vector. The target cells dictating this response and the molecular mechanisms dictating cellular response to AAV are poorly understood. Here, we will investigate the current known AAV capsid and genome interactions with cellular proteins during early stage vector transduction and how these interactions may influence innate cellular responses. We will discuss the current understanding of innate immune activation and DNA damage response to AAV, and the limitations of what is currently known. In particular, we will focus on pathway differences in cell line verses primary cells, with a focus on hematopoietic stem and progenitor cells (HSPCs) in the context of ex-vivo gene editing, and what we can learn from HSPC infection by other parvoviruses. Finally, we will discuss how innate immune and DNA damage response pathway activation in these highly sensitive stem cell populations may impact long-term engraftment and clinical outcomes as these gene-editing strategies move towards the clinic, with the aim to propose pathways relevant for improved hematopoietic stem cell survival and long-term engraftment after AAV-mediated genome editing.},
}

Animals
CRISPR-Cas Systems
Capsid/metabolism
Capsid Proteins/genetics
*DNA Damage
Dependovirus/*genetics/immunology
*Gene Editing
Genetic Therapy
*Genetic Vectors
Genome, Viral
Humans
*Immunity, Innate
Mice

@article {pmid33991533,
year = {2021},
author = {Mardick, JI and Rasmussen, NR and Wightman, B and Reiner, DJ},
title = {Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system.},
journal = {Developmental biology},
volume = {477},
number = {},
pages = {37-48},
pmid = {33991533},
issn = {1095-564X},
support = {P40 OD010440/OD/NIH HHS/United States ; R01 GM121625/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/embryology/*physiology ; Caenorhabditis elegans Proteins/*physiology ; Embryonic Induction ; Genes, ras ; Guanine Nucleotide Exchange Factors/*physiology ; Nervous System/embryology/*physiopathology ; Neurons/physiology ; *Signal Transduction ; ral GTP-Binding Proteins/*physiology ; ras Proteins/genetics/*physiology ; },
abstract = {Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.},
}

Animals
CRISPR-Cas Systems
Caenorhabditis elegans/embryology/*physiology
Caenorhabditis elegans Proteins/*physiology
Embryonic Induction
Genes, ras
Guanine Nucleotide Exchange Factors/*physiology
Nervous System/embryology/*physiopathology
Neurons/physiology
*Signal Transduction
ral GTP-Binding Proteins/*physiology
ras Proteins/genetics/*physiology

@article {pmid33893795,
year = {2021},
author = {Babaei, M and Sartori, L and Karpukhin, A and Abashkin, D and Matrosova, E and Borodina, I},
title = {Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites.},
journal = {FEMS yeast research},
volume = {21},
number = {4},
pages = {},
pmid = {33893795},
issn = {1567-1364},
mesh = {CRISPR-Cas Systems ; Chromosomes, Fungal/genetics ; Industrial Microbiology ; *Metabolic Engineering ; Plasmids ; RNA, Guide ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.},
}

CRISPR-Cas Systems
Chromosomes, Fungal/genetics
Industrial Microbiology
*Metabolic Engineering
Plasmids
RNA, Guide
Saccharomyces cerevisiae/*genetics

@article {pmid33753848,
year = {2021},
author = {Warwick, T and Schulz, MH and Günther, S and Gilsbach, R and Neme, A and Carlberg, C and Brandes, RP and Seuter, S},
title = {A hierarchical regulatory network analysis of the vitamin D induced transcriptome reveals novel regulators and complete VDR dependency in monocytes.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {6518},
pmid = {33753848},
issn = {2045-2322},
mesh = {CCAAT-Enhancer-Binding Proteins/genetics ; CRISPR-Cas Systems/genetics ; Cholecalciferol/genetics/*pharmacology ; Gene Expression Regulation/drug effects ; Gene Knockout Techniques ; Gene Regulatory Networks/drug effects ; Genome, Human/genetics ; Humans ; Monocytes/drug effects/metabolism ; Proto-Oncogene Protein c-ets-1/genetics ; RNA-Seq ; Receptors, Calcitriol/*genetics ; Transcriptome/drug effects/*genetics ; Vitamin D/*genetics/metabolism ; },
abstract = {The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D3 (1,25(OH)2D3). In order to identify pure genomic transcriptional effects of 1,25(OH)2D3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)2D3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)2D3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)2D3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.},
}

CCAAT-Enhancer-Binding Proteins/genetics
CRISPR-Cas Systems/genetics
Cholecalciferol/genetics/*pharmacology
Gene Expression Regulation/drug effects
Gene Knockout Techniques
Gene Regulatory Networks/drug effects
Genome, Human/genetics
Humans
Monocytes/drug effects/metabolism
Proto-Oncogene Protein c-ets-1/genetics
RNA-Seq
Receptors, Calcitriol/*genetics
Transcriptome/drug effects/*genetics
Vitamin D/*genetics/metabolism

@article {pmid33715060,
year = {2021},
author = {Li, H and Yu, H and Du, S and Li, Q},
title = {CRISPR/Cas9 Mediated High Efficiency Knockout of Myosin Essential Light Chain Gene in the Pacific Oyster (Crassostrea Gigas).},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {23},
number = {2},
pages = {215-224},
pmid = {33715060},
issn = {1436-2236},
support = {2018YFD0900200//Key Technology Research and Development Program of Shandong/ ; 31972789//Foundation for Innovative Research Groups of the National Natural Science Foundation of China (CN)/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Crassostrea/*genetics/growth & development ; INDEL Mutation ; Larva/genetics/growth & development ; Muscle Development/*genetics ; Myosin Heavy Chains/metabolism ; Myosin Light Chains/*genetics ; },
abstract = {Pacific oyster (Crassostrea gigas) is one of the most widely cultivated shellfish species in the world. Because of its economic value and complex life cycle involving drastic changes from a free-swimming larva to a sessile juvenile, C. gigas has been used as a model for developmental, environmental, and aquaculture research. However, due to the lack of genetic tools for functional analysis, gene functions associated with biological or economic traits cannot be easily determined. Here, we reported a successful application of CRISPR/Cas9 technology for knockout of myosin essential light chain gene (CgMELC) in C. gigas. C. gigas embryos injected with sgRNAs/Cas9 contained extensive indel mutations at the target sites. The mutant larvae showed defective musculature and reduced motility. In addition, knockout of CgMELC disrupted the expression and patterning of myosin heavy chain positive myofibers in larvae. Together, these data indicate that CgMELC is involved in larval muscle contraction and myogenesis in oyster larvae.},
}

Animals
CRISPR-Cas Systems
Crassostrea/*genetics/growth & development
INDEL Mutation
Larva/genetics/growth & development
Muscle Development/*genetics
Myosin Heavy Chains/metabolism
Myosin Light Chains/*genetics

@article {pmid33705438,
year = {2021},
author = {Ortolano, NA and Romero-Morales, AI and Rasmussen, ML and Bodnya, C and Kline, LA and Joshi, P and Connelly, JP and Rose, KL and Pruett-Miller, SM and Gama, V},
title = {A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.},
journal = {PloS one},
volume = {16},
number = {3},
pages = {e0248000},
pmid = {33705438},
issn = {1932-6203},
support = {T32 HD007502/HD/NICHD NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytochromes c/metabolism ; Gene Editing ; Humans ; Pluripotent Stem Cells/*metabolism ; Proteomics/methods ; *Signal Transduction ; Transferases/*metabolism ; },
abstract = {CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.},
}

CRISPR-Associated Protein 9
CRISPR-Cas Systems
Cytochromes c/metabolism
Gene Editing
Humans
Pluripotent Stem Cells/*metabolism
Proteomics/methods
*Signal Transduction
Transferases/*metabolism

@article {pmid33638992,
year = {2021},
author = {Miki, D and Wang, R and Li, J and Kong, D and Zhang, L and Zhu, JK},
title = {Gene Targeting Facilitated by Engineered Sequence-Specific Nucleases: Potential Applications for Crop Improvement.},
journal = {Plant & cell physiology},
volume = {62},
number = {5},
pages = {752-765},
pmid = {33638992},
issn = {1471-9053},
support = {//Shanghai Science and Technology Innovation Plan/ ; //Chinese Academy of Sciences/ ; },
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; DNA Breaks, Double-Stranded ; Endonucleases/*genetics ; Enhancer Elements, Genetic ; Epigenesis, Genetic ; Gene Editing ; Gene Targeting/*methods ; Plants/*genetics ; Promoter Regions, Genetic ; Protein Engineering/*methods ; },
abstract = {Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world's population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining and are only rarely repaired via error-free homology-directed repair if an appropriate donor template is provided. Gene targeting (GT), i.e. the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.},
}

CRISPR-Cas Systems
Crops, Agricultural/*genetics
DNA Breaks, Double-Stranded
Endonucleases/*genetics
Enhancer Elements, Genetic
Epigenesis, Genetic
Gene Editing
Gene Targeting/*methods
Plants/*genetics
Promoter Regions, Genetic
Protein Engineering/*methods

@article {pmid33560108,
year = {2021},
author = {Tan, SI and Ng, IS},
title = {CRISPRi-Mediated NIMPLY Logic Gate for Fine-Tuning the Whole-Cell Sensing toward Simple Urine Glucose Detection.},
journal = {ACS synthetic biology},
volume = {10},
number = {2},
pages = {412-421},
doi = {10.1021/acssynbio.1c00014},
pmid = {33560108},
issn = {2161-5063},
mesh = {Biosensing Techniques/*methods ; *CRISPR-Cas Systems ; Cellular Reprogramming Techniques/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA-Directed RNA Polymerases/genetics ; Escherichia coli/*genetics ; Glucose/*analysis ; Glycosuria/*diagnosis ; Plasmids/genetics ; Promoter Regions, Genetic ; RNA, Catalytic/genetics ; },
abstract = {Whole-cell biosensors have been regarded as a prominent alternative to chemical and physical biosensors due to their renewability, environmental friendliness, and biocompatibility. However, there is still a lack of noninvasive measurements of urine glucose, which plays a vital role in monitoring the risk of diabetes in the healthcare system, via whole-cell biosensors. In this study, we characterized a glucose-inducible promoter and further enhanced the sensing performance using three genetic effectors, which encompassed ribozyme regulator (RiboJ), clustered regularly interspaced short palindromic repeat interference (CRISPRi), and plasmid-based T7RNA polymerase (PDT7), to develop the noninvasive glucose biosensor by fluorescent signal. As a result, RiboJ increased dynamic range to 2989 au, but declined signal-to-noise (S/N) to 1.59, while CRISPRi-mediated NIMPLY gate intensified both dynamic range to 5720 au and S/N to 4.58. The use of single PDT7 orthogonal with T7 promoter in cells (i.e., P strain) achieved a 44 180 au of dynamic range with S/N at 3.08. By coupling the PDT7 and NIMPLY-mediated CRISPRi, we constructed an optimum PIGAS strain with the highest S/N value of 4.95. Finally, we adopted the synthetic bacteria into a microdevice to afford an integrative and portable system for daily urine glucose inspection, which would be an alternative approach for medical diagnosis in the future.},
}

Biosensing Techniques/*methods
*CRISPR-Cas Systems
Cellular Reprogramming Techniques/*methods
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
DNA-Directed RNA Polymerases/genetics
Escherichia coli/*genetics
Glucose/*analysis
Glycosuria/*diagnosis
Plasmids/genetics
Promoter Regions, Genetic
RNA, Catalytic/genetics

@article {pmid33525637,
year = {2021},
author = {Cetin, R and Quandt, E and Kaulich, M},
title = {Functional Genomics Approaches to Elucidate Vulnerabilities of Intrinsic and Acquired Chemotherapy Resistance.},
journal = {Cells},
volume = {10},
number = {2},
pages = {},
pmid = {33525637},
issn = {2073-4409},
mesh = {Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/*genetics ; Gene Targeting ; *Genomics ; Humans ; Translational Medical Research ; },
abstract = {Drug resistance is a commonly unavoidable consequence of cancer treatment that results in therapy failure and disease relapse. Intrinsic (pre-existing) or acquired resistance mechanisms can be drug-specific or be applicable to multiple drugs, resulting in multidrug resistance. The presence of drug resistance is, however, tightly coupled to changes in cellular homeostasis, which can lead to resistance-coupled vulnerabilities. Unbiased gene perturbations through RNAi and CRISPR technologies are invaluable tools to establish genotype-to-phenotype relationships at the genome scale. Moreover, their application to cancer cell lines can uncover new vulnerabilities that are associated with resistance mechanisms. Here, we discuss targeted and unbiased RNAi and CRISPR efforts in the discovery of drug resistance mechanisms by focusing on first-in-line chemotherapy and their enforced vulnerabilities, and we present a view forward on which measures should be taken to accelerate their clinical translation.},
}

Antineoplastic Agents/pharmacology
CRISPR-Cas Systems/genetics
Drug Resistance, Neoplasm/*genetics
Gene Targeting
*Genomics
Humans
Translational Medical Research

@article {pmid33501828,
year = {2021},
author = {Spasskaya, DS and Kotlov, MI and Lekanov, DS and Tutyaeva, VV and Snezhkina, AV and Kudryavtseva, AV and Karpov, VL and Karpov, DS},
title = {CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii.},
journal = {ACS synthetic biology},
volume = {10},
number = {2},
pages = {297-308},
doi = {10.1021/acssynbio.0c00426},
pmid = {33501828},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA-Binding Proteins/genetics/metabolism ; Debaryomyces/*enzymology/*genetics ; Extremophiles/enzymology/genetics ; Gene Editing/*methods ; Gene Expression Regulation ; Genome, Fungal ; Organisms, Genetically Modified ; Osmoregulation/genetics ; Oxidative Stress/genetics ; Proteasome Endopeptidase Complex/*genetics/metabolism ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Salt Stress/genetics ; Transcription Factors/genetics/metabolism ; Transcription, Genetic ; },
abstract = {The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.},
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
DNA-Binding Proteins/genetics/metabolism
Debaryomyces/*enzymology/*genetics
Extremophiles/enzymology/genetics
Gene Editing/*methods
Gene Expression Regulation
Genome, Fungal
Organisms, Genetically Modified
Osmoregulation/genetics
Oxidative Stress/genetics
Proteasome Endopeptidase Complex/*genetics/metabolism
Saccharomyces cerevisiae/*genetics
Saccharomyces cerevisiae Proteins/genetics/metabolism
Salt Stress/genetics
Transcription Factors/genetics/metabolism
Transcription, Genetic

@article {pmid33464043,
year = {2021},
author = {Standage-Beier, K and Tekel, SJ and Brafman, DA and Wang, X},
title = {Prime Editing Guide RNA Design Automation Using PINE-CONE.},
journal = {ACS synthetic biology},
volume = {10},
number = {2},
pages = {422-427},
pmid = {33464043},
issn = {2161-5063},
support = {R01 GM106081/GM/NIGMS NIH HHS/United States ; R01 GM121698/GM/NIGMS NIH HHS/United States ; R01 GM131405/GM/NIGMS NIH HHS/United States ; R21 AG056706/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Automation ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics ; DNA Breaks, Double-Stranded ; Drosophila melanogaster/genetics ; Gene Editing/*methods ; Genome ; Humans ; Mice ; Oligonucleotides/genetics ; Polymorphism, Single Nucleotide ; RNA Editing/*genetics ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/genetics ; *Software ; Zebrafish/genetics ; },
abstract = {CRISPR-based technologies are paramount in genome engineering and synthetic biology. Prime editing (PE) is a technology capable of installing genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. Prime editing guide RNAs (pegRNAs) simultaneously encode both guide and edit template sequences. They are more design intensive than CRISPR single guide RNAs (sgRNAs). As such, application of PE technology is hindered by the limited throughput of manual pegRNA design. To that end, we designed a software tool, Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE), that enables high-throughput automated design of pegRNAs and prime editing strategies. PINE-CONE translates edit coordinates and sequences into pegRNA designs, accessory guides, and oligonucleotides for facile cloning workflows. To demonstrate PINE-CONE's utility in studying disease-relevant genotypes, we rapidly design a library of pegRNAs targeting Alzheimer's Disease single nucleotide polymorphisms (SNPs). Overall, PINE-CONE will accelerate the application of PEs in synthetic biology and biomedical research.},
}

Animals
Automation
*CRISPR-Cas Systems
Caenorhabditis elegans/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
DNA/genetics
DNA Breaks, Double-Stranded
Drosophila melanogaster/genetics
Gene Editing/*methods
Genome
Humans
Mice
Oligonucleotides/genetics
Polymorphism, Single Nucleotide
RNA Editing/*genetics
RNA, Guide/*genetics
Saccharomyces cerevisiae/genetics
*Software
Zebrafish/genetics

@article {pmid33455878,
year = {2021},
author = {Park, SH and Bao, G},
title = {CRISPR/Cas9 gene editing for curing sickle cell disease.},
journal = {Transfusion and apheresis science : official journal of the World Apheresis Association : official journal of the European Society for Haemapheresis},
volume = {60},
number = {1},
pages = {103060},
pmid = {33455878},
issn = {1473-0502},
support = {R01 HL152314/HL/NHLBI NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*therapy ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; },
abstract = {Sickle cell disease (SCD) is the most common monogenic blood disorder marked by severe pain, end-organ damage, and early mortality. Treatment options for SCD remain very limited. There are only four FDA approved drugs to reduce acute complications. The only curative therapy for SCD is hematopoietic stem cell transplantation, typically from a matched, related donor. Ex vivo engineering of autologous hematopoietic stem and progenitor cells followed by transplantation of genetically modified cells potentially provides a permanent cure applicable to all patients regardless of the availability of suitable donors and graft-vs-host disease. In this review, we focus on the use of CRISPR/Cas9 gene-editing for curing SCD, including the curative correction of SCD mutation in β-globin (HBB) and the induction of fetal hemoglobin to reverse sickling. We summarize the major achievements and challenges, aiming to provide a clearer perspective on the potential of gene-editing based approaches in curing SCD.},
}

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

@article {pmid33420513,
year = {2021},
author = {Houzelstein, D and Simon-Chazottes, D and Batista, L and Tokuda, S and Langa Vives, F and Flamand, M and Montagutelli, X and Panthier, JJ},
title = {The ring finger protein 213 gene (Rnf213) contributes to Rift Valley fever resistance in mice.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {32},
number = {1},
pages = {30-37},
pmid = {33420513},
issn = {1432-1777},
mesh = {Adenosine Triphosphatases/*genetics ; Animals ; CRISPR-Cas Systems ; Chromosome Mapping ; Disease Models, Animal ; Disease Resistance/*genetics ; Host-Pathogen Interactions/*genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Rift Valley Fever/*genetics/*virology ; Rift Valley fever virus/*physiology ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {Rift Valley fever (RVF) is an emerging viral zoonosis that primarily affects ruminants and humans. We have previously shown that wild-derived MBT/Pas mice are highly susceptible to RVF virus and that part of this phenotype is controlled by a locus located on distal Chromosome 11. Using congenic strains, we narrowed down the critical interval to a 530 kb region containing five protein-coding genes among which Rnf213 emerged as a potential candidate. We generated Rnf213-deficient mice by CRISPR/CAS9 on the C57BL/6 J background and showed that they were significantly more susceptible to RVF than control mice, with an average survival time post-infection reduced from 7 to 4 days. The human RNF213 gene had been associated with the cerebrovascular Moyamoya disease (MMD or MYMY) but the inactivation of this gene in the mouse resulted only in mild anomalies of the neovascularization. This study provides the first evidence that the Rnf213 gene may also impact the resistance to infectious diseases such as RVF.},
}

Adenosine Triphosphatases/*genetics
Animals
CRISPR-Cas Systems
Chromosome Mapping
Disease Models, Animal
Disease Resistance/*genetics
Host-Pathogen Interactions/*genetics
Mice
Mice, Inbred C57BL
Mice, Knockout
Rift Valley Fever/*genetics/*virology
Rift Valley fever virus/*physiology
Ubiquitin-Protein Ligases/*genetics

@article {pmid33322954,
year = {2021},
author = {Grand Moursel, L and Visser, M and Servant, G and Durmus, S and Zuurmond, AM},
title = {CRISPRing future medicines.},
journal = {Expert opinion on drug discovery},
volume = {16},
number = {4},
pages = {463-473},
doi = {10.1080/17460441.2021.1850687},
pmid = {33322954},
issn = {1746-045X},
mesh = {Animals ; Biomedical Research/methods ; CRISPR-Cas Systems/*genetics ; Cell- and Tissue-Based Therapy ; Cost-Benefit Analysis ; Drug Development/methods ; Drug Discovery/*methods ; Genetic Engineering ; Genetic Therapy/*methods ; Humans ; },
abstract = {Introduction: The ability to engineer mammalian genomes in a quick and cost-effective way has led to rapid adaptation of CRISPR technology in biomedical research. CRISPR-based engineering has the potential to accelerate drug discovery, to support the reduction of high attrition rate in drug development and to enhance development of cell and gene-based therapies.Areas covered: How CRISPR technology is transforming drug discovery is discussed in this review. From target identification to target validation in both in vitro and in vivo models, CRISPR technology is positively impacting the early stages of drug development by providing a straightforward way to genome engineering. This property also attracted attention for CRISPR application in the cell and gene therapy area.Expert opinion: CRISPR technology is rapidly becoming the preferred tool for genome engineering and nowadays it is hard to imagine the drug discovery pipeline without this technology. With the years to come, CRISPR technology will undoubtedly be further refined and will flourish into a mature technology that will play a key role in supporting genome engineering requirements in the drug discovery pipeline as well as in cell and gene therapy development.},
}

Animals
Biomedical Research/methods
CRISPR-Cas Systems/*genetics
Cell- and Tissue-Based Therapy
Cost-Benefit Analysis
Drug Development/methods
Drug Discovery/*methods
Genetic Engineering
Genetic Therapy/*methods
Humans

@article {pmid33184093,
year = {2020},
author = {Beigl, TB and Kjosås, I and Seljeseth, E and Glomnes, N and Aksnes, H},
title = {Efficient and crucial quality control of HAP1 cell ploidy status.},
journal = {Biology open},
volume = {9},
number = {11},
pages = {},
pmid = {33184093},
issn = {2046-6390},
mesh = {CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Diploidy ; Flow Cytometry ; Gene Editing ; Gene Knockdown Techniques ; Haploidy ; Humans ; Nerve Tissue Proteins/*genetics/metabolism ; *Ploidies ; },
abstract = {The near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.},
}

CRISPR-Cas Systems
Cell Line
Cells, Cultured
Diploidy
Flow Cytometry
Gene Editing
Gene Knockdown Techniques
Haploidy
Humans
Nerve Tissue Proteins/*genetics/metabolism
*Ploidies

@article {pmid34627381,
year = {2021},
author = {Lin, Q and Le, QA and Takebayashi, K and Thongkittidilok, C and Wittayarat, M and Hirata, M and Tanihara, F and Otoi, T},
title = {Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events.},
journal = {BMC research notes},
volume = {14},
number = {1},
pages = {389},
pmid = {34627381},
issn = {1756-0500},
mesh = {Animals ; Blastocyst ; CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide ; Swine ; *Zygote ; },
abstract = {OBJECTIVE: Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection.

RESULTS: Zona pellucida (ZP)-free zygotes collected at 5, 10, and 15 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5 h. Lipofection of zygotes collected at 10 and 15 h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20 h. The blastocyst formation rate of zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required.},
}

Animals
Blastocyst
CRISPR-Cas Systems/genetics
*Gene Editing
RNA, Guide
Swine
*Zygote

@article {pmid34535663,
year = {2021},
author = {Gu, P and Jia, S and Takasugi, T and Tesmer, VM and Nandakumar, J and Chen, Y and Chang, S},
title = {Distinct functions of POT1 proteins contribute to the regulation of telomerase recruitment to telomeres.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5514},
pmid = {34535663},
issn = {2041-1723},
support = {R01 CA202816/CA/NCI NIH HHS/United States ; R01 AG050509/AG/NIA NIH HHS/United States ; R01 GM120094/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Mutational Analysis ; DNA-Binding Proteins/*metabolism ; Mice ; Protein Binding ; Rad51 Recombinase/metabolism ; Sarcoma/pathology ; Telomerase/*metabolism ; Telomere/*metabolism ; Telomere-Binding Proteins/*metabolism ; },
abstract = {Human shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerase-dependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment.},
}

Animals
CRISPR-Cas Systems/genetics
DNA Mutational Analysis
DNA-Binding Proteins/*metabolism
Mice
Protein Binding
Rad51 Recombinase/metabolism
Sarcoma/pathology
Telomerase/*metabolism
Telomere/*metabolism
Telomere-Binding Proteins/*metabolism

@article {pmid34535658,
year = {2021},
author = {Blombach, F and Fouqueau, T and Matelska, D and Smollett, K and Werner, F},
title = {Promoter-proximal elongation regulates transcription in archaea.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5524},
pmid = {34535658},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; WT 207446/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA/metabolism ; DNA-Directed RNA Polymerases/metabolism ; Oxidative Stress/genetics ; *Promoter Regions, Genetic ; Regression Analysis ; Sulfolobus solfataricus/*genetics/growth & development ; *Transcription Elongation, Genetic ; },
abstract = {Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.},
}

CRISPR-Cas Systems/genetics
DNA/metabolism
DNA-Directed RNA Polymerases/metabolism
Oxidative Stress/genetics
*Promoter Regions, Genetic
Regression Analysis
Sulfolobus solfataricus/*genetics/growth & development
*Transcription Elongation, Genetic

@article {pmid33789675,
year = {2021},
author = {Salekdeh, PR and Ma'mani, L and Tavakkoly-Bazzaz, J and Mousavi, H and Modarressi, MH and Salekdeh, GH},
title = {Bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica nanoparticles: a promising nanocarrier for delivery of Cas9-sgRNA ribonucleoproteine.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {95},
pmid = {33789675},
issn = {1477-3155},
mesh = {Adsorption ; CRISPR-Cas Systems ; Cell Survival ; Cloning, Molecular ; Drug Liberation ; Dynamic Light Scattering ; Gene Editing/methods ; Guanidines/*chemistry ; Nanoparticles/*chemistry ; Polyethylene Glycols/*chemistry ; Polymers/chemistry ; RNA, Guide/genetics ; Ribonucleoproteins/*chemistry ; Silanes ; Streptococcus pyogenes/genetics ; },
abstract = {BACKGROUND: There is a great interest in the efficient intracellular delivery of Cas9-sgRNA ribonucleoprotein complex (RNP) and its possible applications for in vivo CRISPR-based gene editing. In this study, a nanoporous mediated gene-editing approach has been successfully performed using a bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica (PMO) nanoparticles (RNP@AGu@PEG1500-PMO) as a potent and biocompatible nanocarrier for RNP delivery.

RESULTS: The bi-functionalized MSN-based nanomaterials have been fully characterized using electron microscopy (TEM and SEM), nitrogen adsorption measurements, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and dynamic light scattering (DLS). The results confirm that AGu@PEG1500-PMO can be applied for gene-editing with an efficiency of about 40% as measured by GFP gene knockdown of HT1080-GFP cells with no notable change in the morphology of the cells.

CONCLUSIONS: Due to the high stability and biocompatibility, simple synthesis, and cost-effectiveness, the developed bi-functionalized PMO-based nano-network introduces a tailored nanocarrier that has remarkable potential as a promising trajectory for biomedical and RNP delivery applications.},
}

Adsorption
CRISPR-Cas Systems
Cell Survival
Cloning, Molecular
Drug Liberation
Dynamic Light Scattering
Gene Editing/methods
Guanidines/*chemistry
Nanoparticles/*chemistry
Polyethylene Glycols/*chemistry
Polymers/chemistry
RNA, Guide/genetics
Ribonucleoproteins/*chemistry
Silanes
Streptococcus pyogenes/genetics

@article {pmid33384170,
year = {2021},
author = {Zheng, X and Kuijer, HNJ and Al-Babili, S},
title = {Carotenoid Biofortification of Crops in the CRISPR Era.},
journal = {Trends in biotechnology},
volume = {39},
number = {9},
pages = {857-860},
doi = {10.1016/j.tibtech.2020.12.003},
pmid = {33384170},
issn = {1879-3096},
mesh = {*Biofortification/trends ; *CRISPR-Cas Systems ; *Carotenoids ; Gene Editing/trends ; *Plants, Genetically Modified/genetics ; },
abstract = {Carotenoids are micronutrients important for human health. The continuous improvements in clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing techniques make rapid, DNA/transgene-free and targeted multiplex genetic modification a reality, thus promising to accelerate the breeding and generation of 'golden' staple crops. We discuss here the progress and future prospects of CRISPR/Cas9 applications for carotenoid biofortification.},
}

*Biofortification/trends
*CRISPR-Cas Systems
*Carotenoids
Gene Editing/trends
*Plants, Genetically Modified/genetics

@article {pmid33268478,
year = {2020},
author = {Alberti, F and Kaleem, S and Weaver, JA},
title = {Recent developments of tools for genome and metabolome studies in basidiomycete fungi and their application to natural product research.},
journal = {Biology open},
volume = {9},
number = {12},
pages = {},
pmid = {33268478},
issn = {2046-6390},
mesh = {Basidiomycota/*genetics/*metabolism ; Biological Products/chemistry/*metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; Genetic Engineering/methods ; *Genomics/methods ; *Metabolomics/methods ; Research ; Secondary Metabolism ; Structure-Activity Relationship ; },
abstract = {Basidiomycota are a large and diverse phylum of fungi. They can make bioactive metabolites that are used or have inspired the synthesis of antibiotics and agrochemicals. Terpenoids are the most abundant class of natural products encountered in this taxon. Other natural product classes have been described, including polyketides, peptides, and indole alkaloids. The discovery and study of natural products made by basidiomycete fungi has so far been hampered by several factors, which include their slow growth and complex genome architecture. Recent developments of tools for genome and metabolome studies are allowing researchers to more easily tackle the secondary metabolome of basidiomycete fungi. Inexpensive long-read whole-genome sequencing enables the assembly of high-quality genomes, improving the scaffold upon which natural product gene clusters can be predicted. CRISPR/Cas9-based engineering of basidiomycete fungi has been described and will have an important role in linking natural products to their genetic determinants. Platforms for the heterologous expression of basidiomycete genes and gene clusters have been developed, enabling natural product biosynthesis studies. Molecular network analyses and publicly available natural product databases facilitate data dereplication and natural product characterisation. These technological advances combined are prompting a revived interest in natural product discovery from basidiomycete fungi.This article has an associated Future Leader to Watch interview with the first author of the paper.},
}

Basidiomycota/*genetics/*metabolism
Biological Products/chemistry/*metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Fungal
Genetic Engineering/methods
*Genomics/methods
*Metabolomics/methods
Research
Secondary Metabolism
Structure-Activity Relationship

@article {pmid33162553,
year = {2020},
author = {Ha, DI and Lee, JM and Lee, NE and Kim, D and Ko, JH and Kim, YS},
title = {Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2'-O-methylated uridinylate-rich 3'-overhang in mouse zygotes.},
journal = {Experimental & molecular medicine},
volume = {52},
number = {11},
pages = {1823-1830},
pmid = {33162553},
issn = {2092-6413},
support = {NRF-2016M3A9B6903343//Ministry of Science, ICT and Future Planning (MSIP)/ ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; },
mesh = {Animals ; Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; Endodeoxyribonucleases/*metabolism ; *Gene Editing/methods ; Genetic Engineering ; Mice ; *RNA, Guide ; Ribose/analogs & derivatives/metabolism ; Substrate Specificity ; Zygote/*metabolism ; },
abstract = {The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3'-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2'-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.},
}

Animals
Bacterial Proteins/*metabolism
CRISPR-Associated Proteins/*metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA Cleavage
Endodeoxyribonucleases/*metabolism
*Gene Editing/methods
Genetic Engineering
Mice
*RNA, Guide
Ribose/analogs & derivatives/metabolism
Substrate Specificity
Zygote/*metabolism