Pub Date : 2023-10-01Epub Date: 2023-09-07DOI: 10.1089/crispr.2023.0033
Kaivalya Molugu, Namita Khajanchi, Cicera R Lazzarotto, Shengdar Q Tsai, Krishanu Saha
Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.
{"title":"Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells.","authors":"Kaivalya Molugu, Namita Khajanchi, Cicera R Lazzarotto, Shengdar Q Tsai, Krishanu Saha","doi":"10.1089/crispr.2023.0033","DOIUrl":"10.1089/crispr.2023.0033","url":null,"abstract":"<p><p>Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"473-485"},"PeriodicalIF":3.7,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10611976/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10307257","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}
Pub Date : 2023-08-01DOI: 10.1089/crispr.2022.0095
Cathryn M Mayes, Joshua L Santarpia
CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.
{"title":"Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.","authors":"Cathryn M Mayes, Joshua L Santarpia","doi":"10.1089/crispr.2022.0095","DOIUrl":"https://doi.org/10.1089/crispr.2022.0095","url":null,"abstract":"<p><p>CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 4","pages":"359-368"},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10457650/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10152767","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}
Pub Date : 2023-08-01DOI: 10.1089/crispr.2023.0032
Daniel F M Monte
{"title":"My CRISPR Story: Back to Brazil.","authors":"Daniel F M Monte","doi":"10.1089/crispr.2023.0032","DOIUrl":"https://doi.org/10.1089/crispr.2023.0032","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 4","pages":"313-315"},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10027836","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}
Pub Date : 2023-08-01DOI: 10.1089/crispr.2023.0006
Allison Sharrar, Luisa Arake de Tacca, Trevor Collingwood, Zuriah Meacham, David Rabuka, Johanna Staples-Ager, Michael Schelle
Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.
{"title":"Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences.","authors":"Allison Sharrar, Luisa Arake de Tacca, Trevor Collingwood, Zuriah Meacham, David Rabuka, Johanna Staples-Ager, Michael Schelle","doi":"10.1089/crispr.2023.0006","DOIUrl":"https://doi.org/10.1089/crispr.2023.0006","url":null,"abstract":"<p><p>Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 4","pages":"350-358"},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10037354","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}
Pub Date : 2023-08-01DOI: 10.1089/crispr.2023.0016
Juliane Weller, Ananth Pallaseni, Jonas Koeppel, Leopold Parts
The first fruits of the CRISPR-Cas revolution are starting to enter the clinic, with gene editing therapies offering solutions to previously incurable genetic diseases. The success of such applications hinges on control over the mutations that are generated, which are known to vary depending on the targeted locus. In this review, we present the current state of understanding and predicting CRISPR-Cas cutting, base editing, and prime editing outcomes in mammalian cells. We first provide an introduction to the basics of DNA repair and machine learning that the models rely on. We then overview the datasets and methods created for characterizing edits at scale, as well as the insights that have been derived from them. The predictions generated from these models serve as a foundation for designing efficient experiments across the broad contexts where these tools are applied.
{"title":"Predicting Mutations Generated by Cas9, Base Editing, and Prime Editing in Mammalian Cells.","authors":"Juliane Weller, Ananth Pallaseni, Jonas Koeppel, Leopold Parts","doi":"10.1089/crispr.2023.0016","DOIUrl":"https://doi.org/10.1089/crispr.2023.0016","url":null,"abstract":"<p><p>The first fruits of the CRISPR-Cas revolution are starting to enter the clinic, with gene editing therapies offering solutions to previously incurable genetic diseases. The success of such applications hinges on control over the mutations that are generated, which are known to vary depending on the targeted locus. In this review, we present the current state of understanding and predicting CRISPR-Cas cutting, base editing, and prime editing outcomes in mammalian cells. We first provide an introduction to the basics of DNA repair and machine learning that the models rely on. We then overview the datasets and methods created for characterizing edits at scale, as well as the insights that have been derived from them. The predictions generated from these models serve as a foundation for designing efficient experiments across the broad contexts where these tools are applied.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 4","pages":"325-338"},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10036531","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}
Pub Date : 2023-08-01DOI: 10.1089/crispr.2023.29162.rfs2022
Nicole F Brackett
{"title":"Rosalind Franklin Society Proudly Announces the 2022 Award Recipient for <i>The CRISPR Journal</i>.","authors":"Nicole F Brackett","doi":"10.1089/crispr.2023.29162.rfs2022","DOIUrl":"https://doi.org/10.1089/crispr.2023.29162.rfs2022","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 4","pages":"401"},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10031407","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}
Pub Date : 2023-08-01DOI: 10.1089/crispr.2022.0096
Yen-Ho Chen, Shakuntala Sharma, William P Bewg, Liang-Jiao Xue, Cole R Gizelbach, Chung-Jui Tsai
The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ∼100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.
CRISPR-Cas9系统已被用于越来越多的物种的精确诱变,包括农作物和森林树木。它在具有极高序列相似性的紧密连接基因中的应用研究较少。在这项研究中,我们使用CRISPR-Cas9诱变了白杨(Populus tremula × Populus alba)中7个NRX1基因的串联阵列,长度约为100 kb。我们在42个转基因品系中展示了使用单个引导RNA的高效多重编辑。突变谱范围从单个基因的小插入和缺失和局部缺失到跨串联基因的大基因组缺失和重排。我们还检测到复杂的重排,包括由多次裂解和修复事件引起的易位和倒位。靶捕获测序对于重建异常突变等位基因的修复结果的公正评估是有用的。这项工作强调了CRISPR-Cas9对串联复制基因进行多重编辑的能力,可以产生具有结构和拷贝数变化的多种突变体,以帮助未来的功能表征。
{"title":"Multiplex Editing of the <i>Nucleoredoxin1</i> Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions.","authors":"Yen-Ho Chen, Shakuntala Sharma, William P Bewg, Liang-Jiao Xue, Cole R Gizelbach, Chung-Jui Tsai","doi":"10.1089/crispr.2022.0096","DOIUrl":"https://doi.org/10.1089/crispr.2022.0096","url":null,"abstract":"<p><p>The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven <i>Nucleoredoxin1</i> (<i>NRX1</i>) genes spanning ∼100 kb in <i>Populus tremula</i> × <i>Populus alba</i>. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 4","pages":"339-349"},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10460964/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10100491","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}
Pub Date : 2023-08-01DOI: 10.1089/crispr.2023.0007
Gabriel Lamothe, Julie Carbonneau, Charles Joly Beauparlant, Thierry Vincent, Patrik Quessy, Anthony Guedon, Gary Kobinger, Jean-Francois Lemay, Guy Boivin, Arnaud Droit, Nathalie Turgeon, Jacques P Tremblay
The worldwide proliferation of the SARS-CoV-2 virus in the past 3 years has allowed the virus to accumulate numerous mutations. Dangerous lineages have emerged one after another, each leading to a new wave of the pandemic. In this study, we have developed the THRASOS pipeline to rapidly discover lineage-specific mutation signatures and thus advise the development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic tests. We also optimized a strategy to modify loop-mediated isothermal amplification amplicons for downstream use with Cas12 and Cas13 for future multiplexing. The close ancestry of the BA.1 and BA.2 variants of SARS-CoV-2 (Omicron) made these excellent candidates for the development of a first test using this workflow. With a quick turnaround time and low requirements for laboratory equipment, the test we have created is ideally suited for settings such as mobile clinics lacking equipment such as Next-Generation Sequencers or Sanger Sequencers and the personnel to run these devices.
{"title":"Rapid and Technically Simple Detection of SARS-CoV-2 Variants Using CRISPR Cas12 and Cas13.","authors":"Gabriel Lamothe, Julie Carbonneau, Charles Joly Beauparlant, Thierry Vincent, Patrik Quessy, Anthony Guedon, Gary Kobinger, Jean-Francois Lemay, Guy Boivin, Arnaud Droit, Nathalie Turgeon, Jacques P Tremblay","doi":"10.1089/crispr.2023.0007","DOIUrl":"https://doi.org/10.1089/crispr.2023.0007","url":null,"abstract":"<p><p>The worldwide proliferation of the SARS-CoV-2 virus in the past 3 years has allowed the virus to accumulate numerous mutations. Dangerous lineages have emerged one after another, each leading to a new wave of the pandemic. In this study, we have developed the THRASOS pipeline to rapidly discover lineage-specific mutation signatures and thus advise the development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic tests. We also optimized a strategy to modify loop-mediated isothermal amplification amplicons for downstream use with Cas12 and Cas13 for future multiplexing. The close ancestry of the BA.1 and BA.2 variants of SARS-CoV-2 (Omicron) made these excellent candidates for the development of a first test using this workflow. With a quick turnaround time and low requirements for laboratory equipment, the test we have created is ideally suited for settings such as mobile clinics lacking equipment such as Next-Generation Sequencers or Sanger Sequencers and the personnel to run these devices.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 4","pages":"369-385"},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10027205","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}