家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是家蚕的重要病原微生物之一,其引发的病毒病会导致家蚕大量死亡,严重威胁蚕桑产业的高质量发展。近年来,基于CRISPR/Cas12a的分子诊断技术因其高灵敏度、高特异性和操作简...家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是家蚕的重要病原微生物之一,其引发的病毒病会导致家蚕大量死亡,严重威胁蚕桑产业的高质量发展。近年来,基于CRISPR/Cas12a的分子诊断技术因其高灵敏度、高特异性和操作简便等优势而受到广泛关注。针对BmNPV的ie-1基因,设计并筛选了多条crRNA,通过优化crRNA组合,成功建立了一种新型的CRISPR/Cas12a系统多重crRNA检测方法。研究结果表明,crRNA3、crRNA8和crRNA9组合使用时检测效果最佳,30 min内通过肉眼观察荧光可在感染BmNPV 24 h的家蚕中肠组织及感染48 h的血液和丝腺组织中检测出BmNPV。该方法无需复杂的核酸扩增步骤,即可实现对BmNPV的快速可视化检测。展开更多
CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To...CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing, we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.展开更多
The CRISPR RNA(crRNA)consists of a conserved repeat RNA(rRNA)and an alterable spacer RNA(sRNA),which can guide the Cas12a effector to recognize and target DNA molecules of interest in both full-length and split fashio...The CRISPR RNA(crRNA)consists of a conserved repeat RNA(rRNA)and an alterable spacer RNA(sRNA),which can guide the Cas12a effector to recognize and target DNA molecules of interest in both full-length and split fashion.We herein demonstrated the split crRNA can be repurposed for RNA detection through serving the sRNA as an RNA target.Inspired by this phenomenon,we developed a Cas12a-based direct RNA detection method,known as split crRNA-motivated amplification-free RNA testing(SMART).We adopted SMART to detect both short-stranded and long-stranded RNA target using two Cas12a orthologs(LbCas12a and FnCas12a),and it showed more prominent ability in detecting short-stranded RNA than long-stranded RNA.The potential mechanism revealed that RNA overhangs impede the RNA strand and dsDNA activator from accessing the catalytic site in the RuvC domain of the Cas12a effector,compromising the stability of the quaternary complex,and thus reducing the efficiency of SMART.Surprisingly,by simply introducing a short DNA activator,SMART could detect attomolar miRNA targets and femtomolar long-stranded RNA target without the need for additional preamplification or reverse transcription procedures.In addition,SMART showed wonderful discrimination ability toward single-nucleotide mutations.Moreover,the collaboration of SMART with the CRISPR–Cas13a system enabled simultaneous detection of multiplex RNAs.Overall,SMART is a simple,yet potent tool that can be flexibly applied to various short-stranded RNA detection,and holds great potential to be extended to other Cas12 orthologs.展开更多
CRISPR‒Cas7-11 is a Type Ⅲ-E CRISPR-associated nuclease that functions as a potent RNA editing tool.Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer(TPR-CHAT)acts as a regulatory protein tha...CRISPR‒Cas7-11 is a Type Ⅲ-E CRISPR-associated nuclease that functions as a potent RNA editing tool.Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer(TPR-CHAT)acts as a regulatory protein that interacts with CRISPR RNA(crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex(Craspase).However,the precise modulation of Cas7-11’s nuclease activity by TPR-CHAT to enhance its utility requires further study.Here,we report cryo-electron microscopy(cryo-EM)structures of Desulfonema ishimotonii(Di)Cas7-11-crRNA,complexed with or without the full length or the N-terminus of TPR-CHAT.These structures unveil the molecular features of the Craspase complex.Structural analysis,combined with in vitro nuclease assay and electrophoretic mobility shift assay,reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT(DiTPR-CHAT_(NTD)).Our work demonstrates that DiTPRCHAT_(NTD) can function as a small unit of DiCas7-11 regulator,potentially enabling safe applications to prevent overcutting and offtarget effects of the CRISPR‒Cas7-11 system.展开更多
CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria.Organisms of Sulfolobales,an order of thermophilic acidophiles belonging to the Cren...CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria.Organisms of Sulfolobales,an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum,usually contain both type I and typeⅢCRISPR-Cas systems.Two species,Saccharolobus solfataricus and Sulfolobus islandicus,have been important models for CRISPR study in archaea,and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps:adaptation,expression and crRNA processing,and interference.Four subtypes of CRISPR-Cas systems are common in these organisms,including I-A,I-D,Ⅲ-B,andⅢ-D.These cas genes form functional modules,e.g.,all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and i Cas modules.Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea,and these methodologies are useful in demonstration of the protospacer-adjacent motif(PAM)-dependent DNA interference by I-A interference modules and multiple interference activities byⅢ-B Cmr systems.Ribonucleoprotein effector complexes have been isolated for SulfolobalesⅢ-B andⅢ-D systems,and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.展开更多
Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system,also known as CRISPR/Cpf1,has been successfully harnessed for genome engineering in many plants,but not in grapevine yet.Here we develope...Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system,also known as CRISPR/Cpf1,has been successfully harnessed for genome engineering in many plants,but not in grapevine yet.Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006(LbCas12a)in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1(TMT1)and dihydroflavonol-4-reductase 1(DFR1)genes in 41B cells.Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells.Heat treatment(34℃)improved the editing efficiencies of CRISPR/LbCas12a system,and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3%to 44.6%and 29.9%to 37.3%after heat treatment,respectively.Moreover,the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing.In addition,genome editing with truncated crRNAs(trucrRNAs)showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis,whereas trucrRNAs with shorter regions of target complementarity≤18 nt in length may not induce detectable mutations in 41B cells.All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.展开更多
基金funded by the National Key Research and Development Program of China(2016YFD0101800)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciencesthe National GMO New Variety Breeding Program of China(2016ZX08011-001)。
文摘CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing, we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.
基金supported by the National Natural Science Foundation of China(22274046,22334005)the Science and Technology Innovation Program of Hunan Province(2022RC1114)Hunan Provincial Innovation Foundation for Postgraduate(CX20220455)。
文摘The CRISPR RNA(crRNA)consists of a conserved repeat RNA(rRNA)and an alterable spacer RNA(sRNA),which can guide the Cas12a effector to recognize and target DNA molecules of interest in both full-length and split fashion.We herein demonstrated the split crRNA can be repurposed for RNA detection through serving the sRNA as an RNA target.Inspired by this phenomenon,we developed a Cas12a-based direct RNA detection method,known as split crRNA-motivated amplification-free RNA testing(SMART).We adopted SMART to detect both short-stranded and long-stranded RNA target using two Cas12a orthologs(LbCas12a and FnCas12a),and it showed more prominent ability in detecting short-stranded RNA than long-stranded RNA.The potential mechanism revealed that RNA overhangs impede the RNA strand and dsDNA activator from accessing the catalytic site in the RuvC domain of the Cas12a effector,compromising the stability of the quaternary complex,and thus reducing the efficiency of SMART.Surprisingly,by simply introducing a short DNA activator,SMART could detect attomolar miRNA targets and femtomolar long-stranded RNA target without the need for additional preamplification or reverse transcription procedures.In addition,SMART showed wonderful discrimination ability toward single-nucleotide mutations.Moreover,the collaboration of SMART with the CRISPR–Cas13a system enabled simultaneous detection of multiplex RNAs.Overall,SMART is a simple,yet potent tool that can be flexibly applied to various short-stranded RNA detection,and holds great potential to be extended to other Cas12 orthologs.
基金supported by the National Key Research and Development Program of China(2021YFA1301900,2021YFA1301203 and 2022YFC2303700 to H.D.and Z.S.)the National Natural Science Foundation of China(31900039 and 32170029 to X.T.,81971974 to H.D.,32222040 and 32070049 to Z.S.)+1 种基金the 1.3.5 Project for Disciplines Excellence of West China Hospital,Sichuan University(ZYYC20021 to H.D.)Tianjin Synthetic Biotechnology Innovation Capacity Improvement Action(TSBICIP-KJGG-008 to Z.S.).
文摘CRISPR‒Cas7-11 is a Type Ⅲ-E CRISPR-associated nuclease that functions as a potent RNA editing tool.Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer(TPR-CHAT)acts as a regulatory protein that interacts with CRISPR RNA(crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex(Craspase).However,the precise modulation of Cas7-11’s nuclease activity by TPR-CHAT to enhance its utility requires further study.Here,we report cryo-electron microscopy(cryo-EM)structures of Desulfonema ishimotonii(Di)Cas7-11-crRNA,complexed with or without the full length or the N-terminus of TPR-CHAT.These structures unveil the molecular features of the Craspase complex.Structural analysis,combined with in vitro nuclease assay and electrophoretic mobility shift assay,reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT(DiTPR-CHAT_(NTD)).Our work demonstrates that DiTPRCHAT_(NTD) can function as a small unit of DiCas7-11 regulator,potentially enabling safe applications to prevent overcutting and offtarget effects of the CRISPR‒Cas7-11 system.
基金grants from the Chinese National Transgenic Science and Technology Program(2019ZX08010003 to QS)the National Natural Science Foundation of China(31771380 to QS)+1 种基金the Qingdao Applied Research Fund for postdoctoral researchers(62450079311107 to ZY)the State Key Laboratory of Microbial Technology and Shandong University。
文摘CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria.Organisms of Sulfolobales,an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum,usually contain both type I and typeⅢCRISPR-Cas systems.Two species,Saccharolobus solfataricus and Sulfolobus islandicus,have been important models for CRISPR study in archaea,and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps:adaptation,expression and crRNA processing,and interference.Four subtypes of CRISPR-Cas systems are common in these organisms,including I-A,I-D,Ⅲ-B,andⅢ-D.These cas genes form functional modules,e.g.,all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and i Cas modules.Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea,and these methodologies are useful in demonstration of the protospacer-adjacent motif(PAM)-dependent DNA interference by I-A interference modules and multiple interference activities byⅢ-B Cmr systems.Ribonucleoprotein effector complexes have been isolated for SulfolobalesⅢ-B andⅢ-D systems,and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.
基金Open access funding provided by Shanghai Jiao Tong Universitysupported by grants from the Strategic Priority Research Program of Chinese Academy of Sciences(XDA24030404-3)+2 种基金the National Natural Science Foundation of China(32001994)the Agricultural Breeding Project of Ningxia Hui Autonomous Region(NXNYYZ20210104)the Youth Innovation Promotion Association CAS(2022078).
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system,also known as CRISPR/Cpf1,has been successfully harnessed for genome engineering in many plants,but not in grapevine yet.Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006(LbCas12a)in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1(TMT1)and dihydroflavonol-4-reductase 1(DFR1)genes in 41B cells.Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells.Heat treatment(34℃)improved the editing efficiencies of CRISPR/LbCas12a system,and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3%to 44.6%and 29.9%to 37.3%after heat treatment,respectively.Moreover,the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing.In addition,genome editing with truncated crRNAs(trucrRNAs)showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis,whereas trucrRNAs with shorter regions of target complementarity≤18 nt in length may not induce detectable mutations in 41B cells.All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.
