CRISPR-Cas technology has revolutionized our ability to understand and engineer organisms,evolving from a singular Cas9 model to a diverse CRISPR toolbox.A critical bottleneck in developing new Cas proteins is identif...CRISPR-Cas technology has revolutionized our ability to understand and engineer organisms,evolving from a singular Cas9 model to a diverse CRISPR toolbox.A critical bottleneck in developing new Cas proteins is identifying protospacer adjacent motif(PAM)sequences.Due to the limitations of experimental methods,bioinformatics approaches have become essential.However,existing PAM prediction programs are limited by the small number of spacers in CRISPR-Cas systems,resulting in low accuracy.To address this,we develop PAMPHLET,a pipeline that uses homology searches to identify additional spacers,significantly increasing the number of spacers up to 18-fold.PAMPHLET is validated on 20 CRISPR-Cas systems and successfully predicts PAM sequences for 18 protospacers.These predictions are further validated using the DocMF platform,which characterizes protein-DNA recognition patterns via next-generation sequencing.The high consistency between PAMPHLET predictions and DocMF results for Cas proteins demonstrates the potential of PAMPHLET to enhance PAM sequence prediction accuracy,expedite the discovery process,and accelerate the development of CRISPR tools.展开更多
In the past several years, the CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated protein) system has been harnessed as an efficient and powerful tool for targeted genome editing i...In the past several years, the CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated protein) system has been harnessed as an efficient and powerful tool for targeted genome editing in different prokaryotic and eukaryotic species. Here, we review the development and application of CRISPR/Cas system in rice, emphasizing different varieties of CRISPR/Cas systems have been applied, strategies for multiplex editing, methods for precise gene insertion and replacement, and efficient systems for base editing and site-specific transcriptional regulation. In addition, the biosecurity of CRISPR/Cas system is also discussed, including transgene-free methods and off-target effects of CRISPR/Cas system. Thus, the development and application of CRISPR/Cas system will have significant impact on functional genomic research and variety improvement in rice.展开更多
The application of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas)can be limited due to a lack of compatible protospacer adjacent motif(PAM)sequences in the DNA reg...The application of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas)can be limited due to a lack of compatible protospacer adjacent motif(PAM)sequences in the DNA regions of interest.Recently,SpRY,a variant of Streptococcus pyogenes Cas9(SpCas9),was reported,which nearly completely fulfils the PAM requirement.Meanwhile,PAMs for Sp RY have not been well addressed.In our previous study,we developed the PAM Definition by Observable Sequence Excision(PAM-DOSE)and green fluorescent protein(GFP)-reporter systems to study PAMs in human cells.Herein,we endeavored to identify the PAMs of SpRY with these two methods.The results indicated that 5’-NRN-3’,5’-NTA-3’,and 5’-NCK-3’could be considered as canonical PAMs.5’-NCA-3’and 5’-NTK-3’may serve as non-priority PAMs.At the same time,PAM of 5’-NYC-3’is not recommended for human cells.These findings provide further insights into the application of SpRY for human genome editing.展开更多
This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequen...This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.展开更多
Since their discovery,CRISPR/Cas systems have significantly expanded the genetic toolbox,aiding in the exploration and enhanced production of natural products across various microbes.Among these,class 2 CRISPR/Cas sys...Since their discovery,CRISPR/Cas systems have significantly expanded the genetic toolbox,aiding in the exploration and enhanced production of natural products across various microbes.Among these,class 2 CRISPR/Cas systems are simpler and more broadly used,but they frequently fail to function effectively in many Streptomyces strains.In this study,we present an engineered class 1 typeⅠCRISPR/Cas system derived from Streptomyces avermitilis,which enables efficient gene editing in phylogenetically distant Streptomyces strains.Through a plasmid interference assay,we identified the effective protospacer adjacent motif as 5′-AAN-3′.Utilizing this system,we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb,with efficiencies exceeding 92%.We further utilized this system to insert DNA fragments into different Streptomyces genomes,facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters.Overall,we established a typeⅠCRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces,known for their rich natural product resources.This is the first report of a gene editing tool developed based on the endogenous class 1 typeⅠCRISPR/Cas system in Streptomyces spp.Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.展开更多
The CRISPR/Cas9 system has been widely applied for plant genome editing.The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif(PAM)sequences in the canonical form NGG and non-canonical NAG.A...The CRISPR/Cas9 system has been widely applied for plant genome editing.The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif(PAM)sequences in the canonical form NGG and non-canonical NAG.Although these PAM sequences are extensively distributed across plant genomes,a broader scope of PAM sequence is required to expand the range of genome editing.Here we report the adoption of three variant enzymes,xCas9,SpCas9-NG and XNG-Cas9,to produce targeted mutation in soybean.Sequencing results determined that xCas9 with the NGG and KGA(contains TGA and GGA)PAMs successfully induces genome editing in soybean genome.SpCas9-NG could recognize NGD(contains NGG,NGA and NGT),RGC(contains AGC and GGC),GAA and GAT PAM sites.In addition,XNG-Cas9 was observed to cleave soybean genomic regions with NGG,GAA and AGY(contains AGC and AGT)PAM.Moreover,off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9(enhanced specificity SpCas9)and exCas9(enhanced specificity xCas9)could improve the specificity of the GGA PAM sequence without reducing on-target editing ficiency.These findings significantly expand the scope of Cas9-mediated genome editing in soybean.展开更多
基金supported by grants from the Foundation for Distinguished Young Talents in Higher Education of Guangdong,China(2024KQNCX157)Our work was also supported in part by the Guangdong Provincial Key Laboratory of Interdisciplinary Research and Application for Data Science,BNU-HKBU United International College(2022B1212010006)+1 种基金in part by the Guangdong Higher Education Upgrading Plan(2021-2025)of“Rushing to the Top,Making Up Shortcomings and Strengthening Special Features”(R0400001-22)Additionally,we acknowledge support from the Zhuhai Basic and Applied Basic ResearchFoundation(2220004002717).
文摘CRISPR-Cas technology has revolutionized our ability to understand and engineer organisms,evolving from a singular Cas9 model to a diverse CRISPR toolbox.A critical bottleneck in developing new Cas proteins is identifying protospacer adjacent motif(PAM)sequences.Due to the limitations of experimental methods,bioinformatics approaches have become essential.However,existing PAM prediction programs are limited by the small number of spacers in CRISPR-Cas systems,resulting in low accuracy.To address this,we develop PAMPHLET,a pipeline that uses homology searches to identify additional spacers,significantly increasing the number of spacers up to 18-fold.PAMPHLET is validated on 20 CRISPR-Cas systems and successfully predicts PAM sequences for 18 protospacers.These predictions are further validated using the DocMF platform,which characterizes protein-DNA recognition patterns via next-generation sequencing.The high consistency between PAMPHLET predictions and DocMF results for Cas proteins demonstrates the potential of PAMPHLET to enhance PAM sequence prediction accuracy,expedite the discovery process,and accelerate the development of CRISPR tools.
基金supported by the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciencesthe National Natural Science Foundation of China (Grant No. 31871703)
文摘In the past several years, the CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated protein) system has been harnessed as an efficient and powerful tool for targeted genome editing in different prokaryotic and eukaryotic species. Here, we review the development and application of CRISPR/Cas system in rice, emphasizing different varieties of CRISPR/Cas systems have been applied, strategies for multiplex editing, methods for precise gene insertion and replacement, and efficient systems for base editing and site-specific transcriptional regulation. In addition, the biosecurity of CRISPR/Cas system is also discussed, including transgene-free methods and off-target effects of CRISPR/Cas system. Thus, the development and application of CRISPR/Cas system will have significant impact on functional genomic research and variety improvement in rice.
基金supported by Lin HE’s Academician Workstation of New Medicine and Clinical Translation(No.18331105)the Program for Basic Science and Technology Cooperation Projects of Wenzhou City(No.H22010011),China。
文摘The application of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas)can be limited due to a lack of compatible protospacer adjacent motif(PAM)sequences in the DNA regions of interest.Recently,SpRY,a variant of Streptococcus pyogenes Cas9(SpCas9),was reported,which nearly completely fulfils the PAM requirement.Meanwhile,PAMs for Sp RY have not been well addressed.In our previous study,we developed the PAM Definition by Observable Sequence Excision(PAM-DOSE)and green fluorescent protein(GFP)-reporter systems to study PAMs in human cells.Herein,we endeavored to identify the PAMs of SpRY with these two methods.The results indicated that 5’-NRN-3’,5’-NTA-3’,and 5’-NCK-3’could be considered as canonical PAMs.5’-NCA-3’and 5’-NTK-3’may serve as non-priority PAMs.At the same time,PAM of 5’-NYC-3’is not recommended for human cells.These findings provide further insights into the application of SpRY for human genome editing.
文摘This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.
基金supported by the National Key Research and Development Program of China(2018YFA0903300)the National Natural Science Foundation of China(32071426)+1 种基金the KeyArea Research and Development Program of Guangdong Province(2020B0303070002)the Haihe Laboratory of Sustainable Chemical Transformations(24HHWCSS00006)。
文摘Since their discovery,CRISPR/Cas systems have significantly expanded the genetic toolbox,aiding in the exploration and enhanced production of natural products across various microbes.Among these,class 2 CRISPR/Cas systems are simpler and more broadly used,but they frequently fail to function effectively in many Streptomyces strains.In this study,we present an engineered class 1 typeⅠCRISPR/Cas system derived from Streptomyces avermitilis,which enables efficient gene editing in phylogenetically distant Streptomyces strains.Through a plasmid interference assay,we identified the effective protospacer adjacent motif as 5′-AAN-3′.Utilizing this system,we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb,with efficiencies exceeding 92%.We further utilized this system to insert DNA fragments into different Streptomyces genomes,facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters.Overall,we established a typeⅠCRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces,known for their rich natural product resources.This is the first report of a gene editing tool developed based on the endogenous class 1 typeⅠCRISPR/Cas system in Streptomyces spp.Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.
基金supported by thegrants from Natural Science Foundation of Jiangxi Province(20171ACB20001)National Natural Science Foundation of China(31800224,31960138 and 31960433).
文摘The CRISPR/Cas9 system has been widely applied for plant genome editing.The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif(PAM)sequences in the canonical form NGG and non-canonical NAG.Although these PAM sequences are extensively distributed across plant genomes,a broader scope of PAM sequence is required to expand the range of genome editing.Here we report the adoption of three variant enzymes,xCas9,SpCas9-NG and XNG-Cas9,to produce targeted mutation in soybean.Sequencing results determined that xCas9 with the NGG and KGA(contains TGA and GGA)PAMs successfully induces genome editing in soybean genome.SpCas9-NG could recognize NGD(contains NGG,NGA and NGT),RGC(contains AGC and GGC),GAA and GAT PAM sites.In addition,XNG-Cas9 was observed to cleave soybean genomic regions with NGG,GAA and AGY(contains AGC and AGT)PAM.Moreover,off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9(enhanced specificity SpCas9)and exCas9(enhanced specificity xCas9)could improve the specificity of the GGA PAM sequence without reducing on-target editing ficiency.These findings significantly expand the scope of Cas9-mediated genome editing in soybean.
