CRISPR Journal最新文献_第9页

Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies. 自动化良好生产规范兼容的CRISPR-Cas9编辑造血干细胞和祖细胞用于β-血红蛋白病的临床治疗

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0086

Guillermo Ureña-Bailén, Milena Block, Tommaso Grandi, Faidra Aivazidou, Jona Quednau, Dariusz Krenz, Alberto Daniel-Moreno, Andrés Lamsfus-Calle, Thomas Epting, Rupert Handgretinger, Stefan Wild, Markus Mezger

{"title":"Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies.","authors":"Guillermo Ureña-Bailén, Milena Block, Tommaso Grandi, Faidra Aivazidou, Jona Quednau, Dariusz Krenz, Alberto Daniel-Moreno, Andrés Lamsfus-Calle, Thomas Epting, Rupert Handgretinger, Stefan Wild, Markus Mezger","doi":"10.1089/crispr.2022.0086","DOIUrl":"https://doi.org/10.1089/crispr.2022.0086","url":null,"abstract":"Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated clinical-scale production of genetically modified hematopoietic stem and progenitor cells (HSPCs) using the CliniMACS Prodigy® system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the BCL11A gene, clustered regularly interspaced short palindromic repeat (CRISPR) target in ongoing clinical trials for β-thalassemia and sickle-cell disease treatment, was disrupted by the CRISPR-Cas9 system simulating a large-scale clinical scenario, yielding 100 million HSPCs with high editing efficiency. In vitro erythroid differentiation and high-performance liquid chromatography analyses corroborated fetal hemoglobin resurgence in edited samples, supporting the feasibility of running the complete process of HSPC gene editing in an automated closed system.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9986018/pdf/crispr.2022.0086.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9334433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity In Vivo in Escherichia coli. 基于CRISPR-dCas9的大肠杆菌RNase III活性测定与筛选系统

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0041

Pricila Hauk, Ryan Weeks, Marc Ostermeier

{"title":"A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity In Vivo in Escherichia coli.","authors":"Pricila Hauk, Ryan Weeks, Marc Ostermeier","doi":"10.1089/crispr.2022.0041","DOIUrl":"https://doi.org/10.1089/crispr.2022.0041","url":null,"abstract":"Ribonuclease III (RNase III) and RNase III-like ribonucleases have a wide range of important functions and are found in all organisms, yet a simple and high-throughput in vivo method for measuring RNase III activity does not exist. Typical methods for measuring RNase III activity rely on in vitro RNA analysis or in vivo methods that are not suitable for high-throughput analysis. In this study, we describe our development of a deactivated Cas9 (dCas9)-based in vivo assay for RNase III activity that utilizes RNase III's cleavage of the 5'-untranslated region (UTR) of its own messenger RNA. The key molecule in the system is a hybrid guide RNA (gRNA) between the 5'-UTR of RNase III and gGFP, a gRNA that works with dCas9 to repress GFP expression. This fusion must be cleaved by RNase III for full GFP repression. Our system uses GFP fluorescence to report on Escherichia coli RNase III activity in culture and on an individual cell basis, making it effective for selecting individual cells through fluorescence-activated cell sorting. Homology between enzymes within the RNase III family suggests this assay might be adapted to measure the activity of other enzymes in the RNase III family such as human Dicer or Drosha.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9321705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

PASTE: The Way Forward for Large DNA Insertions. 大DNA插入的前进之路。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2023.0001

Muhammad Arslan Mahmood, Shahid Mansoor

引用次数: 2

Replication Protein Rep Provides Selective Advantage to Viruses in the Presence of CRISPR-Cas Immunity. 复制蛋白Rep在CRISPR-Cas免疫存在下对病毒提供选择性优势。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0037

Weijia Zhang, Yuvaraj Bhoobalan-Chitty, Xichuan Zhai, Yan Hui, Lars Hestbjerg Hansen, Ling Deng, Xu Peng

{"title":"Replication Protein Rep Provides Selective Advantage to Viruses in the Presence of CRISPR-Cas Immunity.","authors":"Weijia Zhang, Yuvaraj Bhoobalan-Chitty, Xichuan Zhai, Yan Hui, Lars Hestbjerg Hansen, Ling Deng, Xu Peng","doi":"10.1089/crispr.2022.0037","DOIUrl":"https://doi.org/10.1089/crispr.2022.0037","url":null,"abstract":"Anti-Clustered regularly interspaced small palindromic repeat (CRISPR) (Acr) phages cooperate to establish a successful infection in CRISPR-containing host. We report here the selective advantage provided by a replication initiator, Rep, toward cooperative host immunosuppression by viruses encoding Acrs. A rep knockout mutant (Δgp16) of Sulfolobus islandicus rod-shaped virus 2 produced around fourfold less virus in a CRISPR-null host, suggesting that Rep is the major replication initiator. In addition to Rep-dependent replication initiation from the viral genomic termini, we detected Rep-independent replication initiation from nonterminal sites. Intriguingly, despite the presence of Acrs, lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the Δgp16 mutant much more quickly in the CRISPR-containing host than in CRISPR-null host. Despite the nonessentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9328521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Gene Editing Corrects In Vitro a G > A GLB1 Transition from a GM1 Gangliosidosis Patient. 基因编辑纠正GM1神经节脂质病患者G > a GLB1的体外转化

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0045

Delphine Leclerc, Louise Goujon, Sylvie Jaillard, Bénédicte Nouyou, Laurence Cluzeau, Léna Damaj, Christèle Dubourg, Amandine Etcheverry, Thierry Levade, Roseline Froissart, Stéphane Dréano, Xavier Guillory, Leif A Eriksson, Erika Launay, Frédéric Mouriaux, Marc-Antoine Belaud-Rotureau, Sylvie Odent, David Gilot

{"title":"Gene Editing Corrects In Vitro a G > A GLB1 Transition from a GM1 Gangliosidosis Patient.","authors":"Delphine Leclerc, Louise Goujon, Sylvie Jaillard, Bénédicte Nouyou, Laurence Cluzeau, Léna Damaj, Christèle Dubourg, Amandine Etcheverry, Thierry Levade, Roseline Froissart, Stéphane Dréano, Xavier Guillory, Leif A Eriksson, Erika Launay, Frédéric Mouriaux, Marc-Antoine Belaud-Rotureau, Sylvie Odent, David Gilot","doi":"10.1089/crispr.2022.0045","DOIUrl":"https://doi.org/10.1089/crispr.2022.0045","url":null,"abstract":"Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of β-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on β-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9986017/pdf/crispr.2022.0045.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9684128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Acknowledgment of Reviewers 2022. 审稿人致谢2022。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2023.29158.ack

引用次数: 0

Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays. 基于AsCas12a和多导阵列的全基因组CRISPR筛选优化

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0093

Sakina Petiwala, Apexa Modi, Tifani Anton, Erin Murphy, Sabah Kadri, Hengcheng Hu, Charles Lu, Michael J Flister, Daniel Verduzco

{"title":"Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays.","authors":"Sakina Petiwala, Apexa Modi, Tifani Anton, Erin Murphy, Sabah Kadri, Hengcheng Hu, Charles Lu, Michael J Flister, Daniel Verduzco","doi":"10.1089/crispr.2022.0093","DOIUrl":"https://doi.org/10.1089/crispr.2022.0093","url":null,"abstract":"Genomewide loss-of-function (LOF) screening using clustered regularly interspaced short palindromic repeats (CRISPR) has facilitated the discovery of novel gene functions across diverse physiological and pathophysiological systems. A challenge with conventional genomewide CRISPR-Cas9 libraries is the unwieldy size (60,000-120,000 constructs), which is resource intensive and prohibitive in some experimental contexts. One solution to streamlining CRISPR screening is by multiplexing two or more guides per gene on a single construct, which enables functional redundancy to compensate for suboptimal gene knockout by individual guides. In this regard, AsCas12a (Cpf1) and its derivatives, for example, enhanced AsCas12a (enAsCas12a), have enabled multiplexed guide arrays to be specifically and efficiently processed for genome editing. Prior studies have established that multiplexed CRISPR-Cas12a libraries perform comparably to the larger equivalent CRISPR-Cas9 libraries, yet the most efficient CRISPR-Cas12a library design remains unresolved. In this study, we demonstrate that CRISPR-Cas12a genomewide LOF screening performed optimally with three guides arrayed per gene construct and could be adapted to robotic cell culture without noticeable differences in screen performance. Thus, the conclusions from this study provide novel insight to streamlining genomewide LOF screening using CRISPR-Cas12a and robotic cell culture.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9334495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

On- and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice. 水稻CRISPR-Cas12b基因组编辑系统的在靶和脱靶分析

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0072

Filiz Gurel, Yuechao Wu, Changtian Pan, Yanhao Cheng, Gen Li, Tao Zhang, Yiping Qi

{"title":"On- and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice.","authors":"Filiz Gurel, Yuechao Wu, Changtian Pan, Yanhao Cheng, Gen Li, Tao Zhang, Yiping Qi","doi":"10.1089/crispr.2022.0072","DOIUrl":"https://doi.org/10.1089/crispr.2022.0072","url":null,"abstract":"The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency. We targeted EPIDERMAL PATTERNING FACTOR LIKE 9 (OsEPFL9) and GRAIN SIZE 3 (OsGS3) genes with GTTG and ATTC protospacer adjacent motifs, respectively. The use of two Alicyclobacillus acidoterrestris scaffolds (Aac and Aa1.2) significantly increased the frequency of targeted mutagenesis. Next, we performed whole-genome sequencing (WGS) of stably transformed T0 rice plants to assess off-target mutations. WGS analysis revealed background mutations in both coding and noncoding regions with no evidence of sgRNA-dependent off-target activity in edited genomes. We also showed Mendelian segregation of insertion and deletion (indel) mutations in T1 generation. In conclusion, both Aac and Aa1.2 scaffolds provided precise and heritable genome editing in rice.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9698283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3'-Truncated sgRNA. 使用3'-截断sgRNA介导的微型crispr - cas12f1介导的单核苷酸微生物基因组编辑。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-02-01 DOI: 10.1089/crispr.2022.0071

Ho Joung Lee, Hyun Ju Kim, Sang Jun Lee

{"title":"Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3'-Truncated sgRNA.","authors":"Ho Joung Lee, Hyun Ju Kim, Sang Jun Lee","doi":"10.1089/crispr.2022.0071","DOIUrl":"https://doi.org/10.1089/crispr.2022.0071","url":null,"abstract":"The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia coli cells obtained by oligonucleotide-directed genome editing. Although double-, triple-, and quadruple-base substitutions were accurately and efficiently performed in the genome, the performance of single-base editing was poor. To resolve this limitation, we serially truncated the 3'-end of sgRNAs and determined the maximal truncation required to maintain the target DNA cleavage activity of Cas12f1. Negative selection of single-nucleotide-edited cells was efficiently performed with the maximally 3'-truncated sgRNA-Cas12f1 complex in vivo. Moreover, Sanger sequencing showed that the accuracy of single-nucleotide substitution, insertion, and deletion in the microbial genome was improved. These results demonstrated that a truncated sgRNA approach could be widely used for accurate CRISPR-mediated genome editing.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9942177/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9704869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Breaking the Rules. 打破规则。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2022-12-01 DOI: 10.1089/crispr.2022.29157.kd

Kevin Davies

引用次数: 0