CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into...CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into the bacterial cells. Zymomonas mobilis is an ethanologenic bacterium, which encodes a subtype I-F CRISPR-Cas system containing three CRISPR loci and a far distant cas gene cluster. Reverse transcription (RT)-PCR analysis revealed that the CRISPR loci were transcribed on both strands. The Cas proteins were suggested to be expressed based on the previous transcriptomic analysis. Challenging with the invader plasmids containing the artificial protospacer with the protospacer adjacent motif (PAM) of NGG or GG exhibited immune interference activity. However, PAM motif of GG seems more effective than NGG in interference activity. Further, the artificial CRISPR arrays with the spacer sequences targeting to the specific genome sites could also lead to strong immune activity, resulting in almost no transformant grown on the agar plates. It was suggested that bacteria like Z. mobilis ZM4 are lack of the rejoining function to heal the double breakage of genomic DNA made by the CRISPR system. Conclusively, the Type I-F CRISPR-Cas system in Z. mobilis ZM4 is active to functionally defense the invading DNA elements.展开更多
CRISPR-Cas13系统(clustered regularly interspaced short palindromic repeats associated Cas system,CRISPR-Cas)是一种快速、高效、精准的新型RNA编辑工具,具有易于设计、结构简单、操作方便、特异性强的特点。综述了CRISPR-Cas13...CRISPR-Cas13系统(clustered regularly interspaced short palindromic repeats associated Cas system,CRISPR-Cas)是一种快速、高效、精准的新型RNA编辑工具,具有易于设计、结构简单、操作方便、特异性强的特点。综述了CRISPR-Cas13在CRISPR分类系统中的地位、CRISPR-Cas13的结构基础以及作用机制、与其他RNA水平调节方法的比较以及目前的应用前景,以期为相关研究提供参考。展开更多
The bacterium Escherichia coli(E.coli)is one of the most widely used chassis microbes employed for the biosynthesis of numerous valuable chemical compounds.In the past decade,the metabolic engineering of E.coli has un...The bacterium Escherichia coli(E.coli)is one of the most widely used chassis microbes employed for the biosynthesis of numerous valuable chemical compounds.In the past decade,the metabolic engineering of E.coli has undergone significant advances,although further productivity improvements will require extensive genome modification,multi-dimensional regulation,and multiple metabolic-pathway coordination.In this context,clustered regularly interspaced short palindromic repeats(CRISPR),along with CRISPR-associated protein(Cas)and its inactive variant(dCas),have emerged as notable recombination and transcriptional regulation tools that are particularly useful for multiplex metabolic engineering in E.coli.In this review,we briefly describe the CRISPR/Cas9 technology in E.coli,and then summarize the recent advances in CRISPR/dCas9 interference(CRISPRi)systems in E.coli,particularly the strategies designed to effectively regulate gene repression and overcome retroactivity during multiplexing.Moreover,we discuss recent applications of the CRISPRi system for enhancing metabolite production in E.coli,and finally highlight the major challenges and future perspectives of this technology.展开更多
文摘CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into the bacterial cells. Zymomonas mobilis is an ethanologenic bacterium, which encodes a subtype I-F CRISPR-Cas system containing three CRISPR loci and a far distant cas gene cluster. Reverse transcription (RT)-PCR analysis revealed that the CRISPR loci were transcribed on both strands. The Cas proteins were suggested to be expressed based on the previous transcriptomic analysis. Challenging with the invader plasmids containing the artificial protospacer with the protospacer adjacent motif (PAM) of NGG or GG exhibited immune interference activity. However, PAM motif of GG seems more effective than NGG in interference activity. Further, the artificial CRISPR arrays with the spacer sequences targeting to the specific genome sites could also lead to strong immune activity, resulting in almost no transformant grown on the agar plates. It was suggested that bacteria like Z. mobilis ZM4 are lack of the rejoining function to heal the double breakage of genomic DNA made by the CRISPR system. Conclusively, the Type I-F CRISPR-Cas system in Z. mobilis ZM4 is active to functionally defense the invading DNA elements.
文摘CRISPR-Cas13系统(clustered regularly interspaced short palindromic repeats associated Cas system,CRISPR-Cas)是一种快速、高效、精准的新型RNA编辑工具,具有易于设计、结构简单、操作方便、特异性强的特点。综述了CRISPR-Cas13在CRISPR分类系统中的地位、CRISPR-Cas13的结构基础以及作用机制、与其他RNA水平调节方法的比较以及目前的应用前景,以期为相关研究提供参考。
基金supported by the Agency for Science,Technology and Research(A∗STAR)under Central Research Fund(Applied/Translational Research),AME Young Individual.Research Grant:A2084c0064(2019).
文摘The bacterium Escherichia coli(E.coli)is one of the most widely used chassis microbes employed for the biosynthesis of numerous valuable chemical compounds.In the past decade,the metabolic engineering of E.coli has undergone significant advances,although further productivity improvements will require extensive genome modification,multi-dimensional regulation,and multiple metabolic-pathway coordination.In this context,clustered regularly interspaced short palindromic repeats(CRISPR),along with CRISPR-associated protein(Cas)and its inactive variant(dCas),have emerged as notable recombination and transcriptional regulation tools that are particularly useful for multiplex metabolic engineering in E.coli.In this review,we briefly describe the CRISPR/Cas9 technology in E.coli,and then summarize the recent advances in CRISPR/dCas9 interference(CRISPRi)systems in E.coli,particularly the strategies designed to effectively regulate gene repression and overcome retroactivity during multiplexing.Moreover,we discuss recent applications of the CRISPRi system for enhancing metabolite production in E.coli,and finally highlight the major challenges and future perspectives of this technology.
