Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in th...Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.展开更多
Genome rearrangement is an important process that leads to genetic diversity,including mutation-related insertions,deletions,or inversions in the genome[1,2].
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various organisms.With efficient...The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various organisms.With efficient and proper expression of multiple guide RNAs(gRNAs),the CRISPR/Cas system is particularly suitable for multiplex genome editing.During the past several years,different CRISPR/Cas expression strategies,such as two-component transcriptional unit,single transcriptional unit,and bidirectional promoter systems,have been developed to efficiently express gRNAs as well as Cas nucleases.Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes,endogenous RNases,and exogenous endoribonuclease(Csy4).Besides being constitutively and ubiquitously expressed,inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible,tissue-specific,and/or synthetic promoters for specific research purposes.Most recently,the emergence of CRISPR/Cas ribonucleoprotein delivery methods,such as engineered nanoparticles,further revolutionized transgene-free and multiplex genome editing.In this review,we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.展开更多
Box C/D RNAs guide the site-specific formation of 2′-O-methylated nucleotides(Nm)of RNAs in eukaryotes and archaea.Although C/D RNAs have been profiled in several archaea,their targets have not been experimentally de...Box C/D RNAs guide the site-specific formation of 2′-O-methylated nucleotides(Nm)of RNAs in eukaryotes and archaea.Although C/D RNAs have been profiled in several archaea,their targets have not been experimentally determined.Here,we mapped Nm in r RNAs,t RNAs,and abundant small RNAs(s RNAs)and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus.The targets of C/D RNAs were assigned by analysis of base-pairing interactions,in vitro modification assays,and gene deletion experiments,revealing a complicated landscape of C/D RNA-target interactions.C/D RNAs widely use dual antisense elements to target adjacent sites in r RNAs,enhancing modification at weakly bound sites.Two consecutive sites can be guided with the same antisense element upstream of box D or D′,a phenomenon known as doublespecificity that is exclusive to internal box D′in eukaryotic C/D RNAs.Several C/D RNAs guide modification at a single non-canonical site.This study reveals the global landscape of RNA-guided 2′-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA.展开更多
The CRISPR-Cas revolution is taking place in virtually all fields of life sciences.Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient,re...The CRISPR-Cas revolution is taking place in virtually all fields of life sciences.Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient,relying only on the design of a synthetic single guide RNA（sgRNA） and its co-expression with Cas9.Here,we review the progress in the design of sgRNA from the original dual RNA guide for S.pyogenes and Staphylococcus aureus Cas9（SpCas9 and SaCas9）.New assays for genome-wide identification of offtargets have provided important insights into the issue of cleavage specificity in vivo.At the same time,the on-target activity of thousands of guides has been determined.These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences.It appears that for most basic research applications,cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions.Moreover,recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing.Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA,several variants of SpCas9 have recently been engineered,either with novel protospacer adjacent motifs（PAMs） or with drastically reduced off-targets.Novel Cas9 and Cas9-like proteins called Cpf 1 have also been characterized from other bacteria and will benefit from die insights obtained from SpCas9.Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.展开更多
Clustered regularly interspaced short palindromic repeat(CRISPR)technologies have opened new scientific avenues widely used in biomedical research.But simple and efficient strategies to reversibly control CRISPR are l...Clustered regularly interspaced short palindromic repeat(CRISPR)technologies have opened new scientific avenues widely used in biomedical research.But simple and efficient strategies to reversibly control CRISPR are lacking.In contrast to previous methods of attaching molecules to the ribose of guide RNAs(gRNAs),we focused on molecules that can directly react with nucleobases.Here,we developed a new strategy to switch off the CRISPR system by efficiently installing 4-(bromomethyl)phenylboronic acid onto nucleobases in gRNAs.CRISPR can then be activated by hydrogen peroxide(H_(2)O_(2)).Collectively,this work demonstrates boronic acid reversibly modulating CRISPR systems through a H_(2)O_(2)-responsive manner.展开更多
CRISPR/Cas9 is a versatile genome editing tool that has the potential to be used to cure many genetic diseases.The system works via a guide RNA(gRNA)interacting with the Cas9 protein to form a complex that binds to a ...CRISPR/Cas9 is a versatile genome editing tool that has the potential to be used to cure many genetic diseases.The system works via a guide RNA(gRNA)interacting with the Cas9 protein to form a complex that binds to a specific DNA sequence.1 The site-specific DNA binding feature of the Cas9 system can be utilized in a variety of ways to correct gene mutations or to regulate gene expression.First,the Cas9 protein can make a site-specific double-stranded break that is mainly repaired by homology-directed repair or non-homologous end-joining.展开更多
CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology,allowing the targeted engineering of a wide range of genomes.The system originally evolved in prokaryotes as an adaptive imm...CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology,allowing the targeted engineering of a wide range of genomes.The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections.It now sees widespread application in genome engineering workflows,especially using the Streptococcus pyogenes endonuclease Cas9.To utilize Cas9,so-called single guide RNAs(sgRNAs)need to be designed for each target gene.While there are many tools available to design sgRNAs for the popular model organisms,only few tools that allow designing sgRNAs for non-model organisms exist.Here,we present CRISPy-web(http://crispy.secondarymetabolites.org/),an easy to use web tool based on CRISPy to design sgRNAs for any userprovided microbial genome.CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs.After checks for potential off-target matches,the resulting sgRNA sequences are displayed graphically and can be exported to text files.All steps and information are accessible from a web browser without the requirement to install and use command line scripts.展开更多
Comprehensive Summary Currently,CRISPR/Cas9 technology has found widespread applications across various domains.However,the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety,prima...Comprehensive Summary Currently,CRISPR/Cas9 technology has found widespread applications across various domains.However,the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety,primarily stemming from the uncontrolled activity of the system.Therefore,the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance.Biotin,characterized by its small molecular weight,and streptavidin,distinguished by its substantial spatial steric hindrance,can be harnessed as an ideal OFF switch(termed a"bioactivity brake")due to their interaction characteristics.In this work,we present a strategy that employs the streptavidin-biotin interaction as a"brake system"for CRISPR/Cas9,effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9.展开更多
基金supported by a National Transgenic Science and Technology Program (2016ZX08010002)to R.W.a startup fund from the Huazhong Agricultural University
文摘Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.
基金supported by grants(92168103,32171417,2019CXJQ01)from the National Nature Science Foundation of China,Shanghai Municipal GovernmentPeak Disciplines(Type IV)of Institutions of Higher Learning in Shanghai.
文摘Genome rearrangement is an important process that leads to genetic diversity,including mutation-related insertions,deletions,or inversions in the genome[1,2].
基金This work was supported by NSF Plant Genome Research Project Grant(1740874)the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04659 and Accession#1016432.
文摘The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various organisms.With efficient and proper expression of multiple guide RNAs(gRNAs),the CRISPR/Cas system is particularly suitable for multiplex genome editing.During the past several years,different CRISPR/Cas expression strategies,such as two-component transcriptional unit,single transcriptional unit,and bidirectional promoter systems,have been developed to efficiently express gRNAs as well as Cas nucleases.Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes,endogenous RNases,and exogenous endoribonuclease(Csy4).Besides being constitutively and ubiquitously expressed,inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible,tissue-specific,and/or synthetic promoters for specific research purposes.Most recently,the emergence of CRISPR/Cas ribonucleoprotein delivery methods,such as engineered nanoparticles,further revolutionized transgene-free and multiplex genome editing.In this review,we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.
基金supported by the Strategic Priority Research Program of Chinese Academy of Sciences(XDB0570000,XDB37010201)the Basic Research Program Based on Major Scientific Infrastructures of Chinese Academy of Sciences(JZHKYPT-2021-05)+1 种基金the National Natural Science Foundation of China(91940302,91540201,31430024,31325007)the National Key Research and Development Program of China(2017YFA0504600)。
文摘Box C/D RNAs guide the site-specific formation of 2′-O-methylated nucleotides(Nm)of RNAs in eukaryotes and archaea.Although C/D RNAs have been profiled in several archaea,their targets have not been experimentally determined.Here,we mapped Nm in r RNAs,t RNAs,and abundant small RNAs(s RNAs)and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus.The targets of C/D RNAs were assigned by analysis of base-pairing interactions,in vitro modification assays,and gene deletion experiments,revealing a complicated landscape of C/D RNA-target interactions.C/D RNAs widely use dual antisense elements to target adjacent sites in r RNAs,enhancing modification at weakly bound sites.Two consecutive sites can be guided with the same antisense element upstream of box D or D′,a phenomenon known as doublespecificity that is exclusive to internal box D′in eukaryotic C/D RNAs.Several C/D RNAs guide modification at a single non-canonical site.This study reveals the global landscape of RNA-guided 2′-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA.
基金funded by ANR(ANR-II-INBS-0014)funded by grants NIH/NHGRI 5U41HG002371-15,NIH/NCI 5U54HG007990-02a grant from the California Institute of Regenerative Medicine,CIRM GC1R-06673C
文摘The CRISPR-Cas revolution is taking place in virtually all fields of life sciences.Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient,relying only on the design of a synthetic single guide RNA（sgRNA） and its co-expression with Cas9.Here,we review the progress in the design of sgRNA from the original dual RNA guide for S.pyogenes and Staphylococcus aureus Cas9（SpCas9 and SaCas9）.New assays for genome-wide identification of offtargets have provided important insights into the issue of cleavage specificity in vivo.At the same time,the on-target activity of thousands of guides has been determined.These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences.It appears that for most basic research applications,cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions.Moreover,recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing.Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA,several variants of SpCas9 have recently been engineered,either with novel protospacer adjacent motifs（PAMs） or with drastically reduced off-targets.Novel Cas9 and Cas9-like proteins called Cpf 1 have also been characterized from other bacteria and will benefit from die insights obtained from SpCas9.Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.
基金the National Natural Science Foundation of China(grant nos.22177089,91853119,21721005,91753201,21877086,and 22177088)the Hubei Natural Science Foundation for Distinguished Young Scholars(grant no.2019CFA064)the Fundamental Research Funds for the Central Universities(grant no.2042019-kf0189).
文摘Clustered regularly interspaced short palindromic repeat(CRISPR)technologies have opened new scientific avenues widely used in biomedical research.But simple and efficient strategies to reversibly control CRISPR are lacking.In contrast to previous methods of attaching molecules to the ribose of guide RNAs(gRNAs),we focused on molecules that can directly react with nucleobases.Here,we developed a new strategy to switch off the CRISPR system by efficiently installing 4-(bromomethyl)phenylboronic acid onto nucleobases in gRNAs.CRISPR can then be activated by hydrogen peroxide(H_(2)O_(2)).Collectively,this work demonstrates boronic acid reversibly modulating CRISPR systems through a H_(2)O_(2)-responsive manner.
基金supported by the New Frontiers in Research Fund(NFRF),administered by the Social Sciences and Humanities Research Council(SSHRC)on behalf of CIHR,NSERC,and SSHRC(Grant#NFRFE-2021-00713).
文摘CRISPR/Cas9 is a versatile genome editing tool that has the potential to be used to cure many genetic diseases.The system works via a guide RNA(gRNA)interacting with the Cas9 protein to form a complex that binds to a specific DNA sequence.1 The site-specific DNA binding feature of the Cas9 system can be utilized in a variety of ways to correct gene mutations or to regulate gene expression.First,the Cas9 protein can make a site-specific double-stranded break that is mainly repaired by homology-directed repair or non-homologous end-joining.
文摘CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology,allowing the targeted engineering of a wide range of genomes.The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections.It now sees widespread application in genome engineering workflows,especially using the Streptococcus pyogenes endonuclease Cas9.To utilize Cas9,so-called single guide RNAs(sgRNAs)need to be designed for each target gene.While there are many tools available to design sgRNAs for the popular model organisms,only few tools that allow designing sgRNAs for non-model organisms exist.Here,we present CRISPy-web(http://crispy.secondarymetabolites.org/),an easy to use web tool based on CRISPy to design sgRNAs for any userprovided microbial genome.CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs.After checks for potential off-target matches,the resulting sgRNA sequences are displayed graphically and can be exported to text files.All steps and information are accessible from a web browser without the requirement to install and use command line scripts.
基金the National Natural Science Foundation of China(Nos.22177089,21721005,92153303,22037004,22177088)the Fundamental Research Funds for the Central Universities(2042023kf0204)Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University(Grant No.ZNJC202309).
文摘Comprehensive Summary Currently,CRISPR/Cas9 technology has found widespread applications across various domains.However,the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety,primarily stemming from the uncontrolled activity of the system.Therefore,the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance.Biotin,characterized by its small molecular weight,and streptavidin,distinguished by its substantial spatial steric hindrance,can be harnessed as an ideal OFF switch(termed a"bioactivity brake")due to their interaction characteristics.In this work,we present a strategy that employs the streptavidin-biotin interaction as a"brake system"for CRISPR/Cas9,effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9.
