Pub Date : 2023-04-01DOI: 10.1089/crispr.2022.0048
Maturada Patchsung, Aimorn Homchan, Kanokpol Aphicho, Surased Suraritdechachai, Thanyapat Wanitchanon, Archiraya Pattama, Khomkrit Sappakhaw, Piyachat Meesawat, Thanakrit Wongsatit, Artittaya Athipanyasilp, Krittapas Jantarug, Niracha Athipanyasilp, Juthamas Buahom, Supapat Visanpattanasin, Nootaree Niljianskul, Pimchai Chaiyen, Ruchanok Tinikul, Nuanjun Wichukchinda, Surakameth Mahasirimongkol, Rujipas Sirijatuphat, Nasikarn Angkasekwinai, Michael A Crone, Paul S Freemont, Julia Joung, Alim Ladha, Omar Abudayyeh, Jonathan Gootenberg, Feng Zhang, Claire Chewapreecha, Sittinan Chanarat, Navin Horthongkham, Danaya Pakotiprapha, Chayasith Uttamapinant
Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.
{"title":"A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.","authors":"Maturada Patchsung, Aimorn Homchan, Kanokpol Aphicho, Surased Suraritdechachai, Thanyapat Wanitchanon, Archiraya Pattama, Khomkrit Sappakhaw, Piyachat Meesawat, Thanakrit Wongsatit, Artittaya Athipanyasilp, Krittapas Jantarug, Niracha Athipanyasilp, Juthamas Buahom, Supapat Visanpattanasin, Nootaree Niljianskul, Pimchai Chaiyen, Ruchanok Tinikul, Nuanjun Wichukchinda, Surakameth Mahasirimongkol, Rujipas Sirijatuphat, Nasikarn Angkasekwinai, Michael A Crone, Paul S Freemont, Julia Joung, Alim Ladha, Omar Abudayyeh, Jonathan Gootenberg, Feng Zhang, Claire Chewapreecha, Sittinan Chanarat, Navin Horthongkham, Danaya Pakotiprapha, Chayasith Uttamapinant","doi":"10.1089/crispr.2022.0048","DOIUrl":"https://doi.org/10.1089/crispr.2022.0048","url":null,"abstract":"<p><p>Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7614457/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9384149","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 : 2023-04-01Epub Date: 2023-03-21DOI: 10.1089/crispr.2022.0074
Melissa D Morales-Moreno, Erick G Valdés-Galindo, Mariana M Reza, Tatiana Fiordelisio, Jorge Peon, Armando Hernandez-Garcia
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.
{"title":"Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.","authors":"Melissa D Morales-Moreno, Erick G Valdés-Galindo, Mariana M Reza, Tatiana Fiordelisio, Jorge Peon, Armando Hernandez-Garcia","doi":"10.1089/crispr.2022.0074","DOIUrl":"10.1089/crispr.2022.0074","url":null,"abstract":"<p><p>Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9330781","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 : 2023-04-01DOI: 10.1089/crispr.2022.0070
Grégoire Cullot, Samuel Amintas, Laura Karembé, Valérie Prouzet-Mauléon, Julie Rébillard, Lisa Boureau, David Cappellen, Aurélie Bedel, François Moreau-Gaudry, Stéphanie Dulucq, Sandrine Dabernat, Béatrice Turcq
Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.
{"title":"Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic <i>BCR::ABL1</i> and <i>EGFR</i> Rearrangements.","authors":"Grégoire Cullot, Samuel Amintas, Laura Karembé, Valérie Prouzet-Mauléon, Julie Rébillard, Lisa Boureau, David Cappellen, Aurélie Bedel, François Moreau-Gaudry, Stéphanie Dulucq, Sandrine Dabernat, Béatrice Turcq","doi":"10.1089/crispr.2022.0070","DOIUrl":"https://doi.org/10.1089/crispr.2022.0070","url":null,"abstract":"<p><p>Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of <i>BCR::ABL1</i> mRNA, a hallmark of chronic myeloid leukemia (CML), and <i>EGFR</i> DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of <i>BCR::ABL1</i> and <i>EGFR</i> alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9699854","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 : 2023-02-20DOI: 10.1101/2022.08.26.505498
Yen-Ho Chen, Shakuntala Sharma, W. 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. Here, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula × alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines. The mutation profiles ranged from small indels 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 functional characterization.
{"title":"Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions","authors":"Yen-Ho Chen, Shakuntala Sharma, W. P. Bewg, Liang-Jiao Xue, Cole R. Gizelbach, Chung-Jui Tsai","doi":"10.1101/2022.08.26.505498","DOIUrl":"https://doi.org/10.1101/2022.08.26.505498","url":null,"abstract":"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. Here, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula × alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines. The mutation profiles ranged from small indels 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 functional characterization.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49032740","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 : 2023-02-01DOI: 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
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.
{"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":"<p><p>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<sup>®</sup> system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the <i>BCL11A</i> 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. <i>In vitro</i> 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.</p>","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}
Pub Date : 2023-02-01DOI: 10.1089/crispr.2022.0041
Pricila Hauk, Ryan Weeks, Marc Ostermeier
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.
{"title":"A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity <i>In Vivo</i> in <i>Escherichia coli</i>.","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":"<p><p>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 <i>in vivo</i> method for measuring RNase III activity does not exist. Typical methods for measuring RNase III activity rely on <i>in vitro</i> RNA analysis or <i>in vivo</i> methods that are not suitable for high-throughput analysis. In this study, we describe our development of a deactivated Cas9 (dCas9)-based <i>in vivo</i> 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 <i>Escherichia coli</i> 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.</p>","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}
Pub Date : 2023-02-01DOI: 10.1089/crispr.2023.0001
Muhammad Arslan Mahmood, Shahid Mansoor
{"title":"PASTE: The Way Forward for Large DNA Insertions.","authors":"Muhammad Arslan Mahmood, Shahid Mansoor","doi":"10.1089/crispr.2023.0001","DOIUrl":"https://doi.org/10.1089/crispr.2023.0001","url":null,"abstract":"","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":"9455443","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 : 2023-02-01DOI: 10.1089/crispr.2022.0037
Weijia Zhang, Yuvaraj Bhoobalan-Chitty, Xichuan Zhai, Yan Hui, Lars Hestbjerg Hansen, Ling Deng, Xu Peng
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.
{"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":"<p><p>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 <i>rep</i> knockout mutant (Δ<i>gp16</i>) of <i>Sulfolobus islandicus</i> 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 (<i>rep</i>-containing) outcompeted the Δ<i>gp16</i> mutant much more quickly in the CRISPR-containing host than in CRISPR-null host. Despite the nonessentiality, <i>rep</i> is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.</p>","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}