Well-established targeted technologies to engi- neer genomes such as zinc-finger nuclease-based editing （ZFN）, transcription activator-like effector nuclease-based editing （TALEN）, and clustered regularly interspa...Well-established targeted technologies to engi- neer genomes such as zinc-finger nuclease-based editing （ZFN）, transcription activator-like effector nuclease-based editing （TALEN）, and clustered regularly interspaced short palindromic repeats and associated protein system-based editing （CRISPR/Cas） are proving to advance basic and applied research in numerous plant species. Compared with systems using ZFNs and TALENs, the most recently developed CRISPR/Cas system is more efficient due to its use of an RNA-guided nuclease to generate double-strand DNA breaks. To accelerate the applications of these technologies, we provide here a detailed overview of these systems, highlight the strengths and weaknesses of each, summarize research advances made with these technologies in model and crop plants, and discuss their applications in plant functional genomics. Such targeted approaches for genetically modifying plants will benefit agricultural production in the future.展开更多
Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexp...Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More importantly,we demonstrated that both ChCas12b and its high-fidelity variant,ChCas12b-D496A,enabled allelespecific disruption of genes harboring single nucleotide polymorphisms(SNPs).These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.展开更多
The human gut microbiome,a complex ecosystem,significantly influences host health,impacting crucial aspects such as metabolism and immunity.To enhance our comprehension and control of the molecular mechanisms orchestr...The human gut microbiome,a complex ecosystem,significantly influences host health,impacting crucial aspects such as metabolism and immunity.To enhance our comprehension and control of the molecular mechanisms orchestrating the intricate interplay between gut commensal bacteria and human health,the exploration of genome engineering for gut microbes is a promising frontier.Nevertheless,the complexities and diversities inherent in the gut microbiome pose substantial challenges to the development of effective genome engineering tools for human gut microbes.In this comprehensive review,we provide an overview of the current progress and challenges in genome engineering of human gut commensal bacteria,whether executed in vitro or in situ.A specific focus is directed towards the advancements and prospects in cargo DNA delivery and high-throughput techniques.Additionally,we elucidate the immense potential of genome engineering methods to enhance our understanding of the human gut microbiome and engineer the microorganisms to enhance human health.展开更多
CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRN...CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.展开更多
文摘Well-established targeted technologies to engi- neer genomes such as zinc-finger nuclease-based editing （ZFN）, transcription activator-like effector nuclease-based editing （TALEN）, and clustered regularly interspaced short palindromic repeats and associated protein system-based editing （CRISPR/Cas） are proving to advance basic and applied research in numerous plant species. Compared with systems using ZFNs and TALENs, the most recently developed CRISPR/Cas system is more efficient due to its use of an RNA-guided nuclease to generate double-strand DNA breaks. To accelerate the applications of these technologies, we provide here a detailed overview of these systems, highlight the strengths and weaknesses of each, summarize research advances made with these technologies in model and crop plants, and discuss their applications in plant functional genomics. Such targeted approaches for genetically modifying plants will benefit agricultural production in the future.
基金supported by the National Key Research and Development Program of China(2021YFC2701103,2021YFA0910602,and 2019YFA0802804)the National Natural Science Foundation of China(82070258 and 31925011)+1 种基金Open Research Fund of State Key Laboratory of Genetic Engineering,Fudan University(SKLGE-2104)Science and Technology Research Program of Shanghai(19DZ2282100)。
文摘Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More importantly,we demonstrated that both ChCas12b and its high-fidelity variant,ChCas12b-D496A,enabled allelespecific disruption of genes harboring single nucleotide polymorphisms(SNPs).These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.
基金National Key R&D Program of China(2019YFA0906700)Guangdong Basic and Applied Basic Research Foundation(2020A1515110184)+1 种基金Dr.Neher's Biophysics Laboratory for Innovative Drug Discovery(001/2020/ALC),regular grants(0056/2020/AMJ&0063/2022/A2)from Macao Science and Technology Development Fund2020 Young Qihuang Scholar funded by National Administration of Traditional Chinese Medicine and also financially supported by the Start-up Research Grant of University of Macao(SRG2022-00020-FHS)and the Faculty of Health Sciences,University of Macao.
文摘The human gut microbiome,a complex ecosystem,significantly influences host health,impacting crucial aspects such as metabolism and immunity.To enhance our comprehension and control of the molecular mechanisms orchestrating the intricate interplay between gut commensal bacteria and human health,the exploration of genome engineering for gut microbes is a promising frontier.Nevertheless,the complexities and diversities inherent in the gut microbiome pose substantial challenges to the development of effective genome engineering tools for human gut microbes.In this comprehensive review,we provide an overview of the current progress and challenges in genome engineering of human gut commensal bacteria,whether executed in vitro or in situ.A specific focus is directed towards the advancements and prospects in cargo DNA delivery and high-throughput techniques.Additionally,we elucidate the immense potential of genome engineering methods to enhance our understanding of the human gut microbiome and engineer the microorganisms to enhance human health.
文摘CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.
