多重基因组编辑中CRISPR-Cas9系统和CRISPR-Cpf1系统的应用和比较

Application and Comparison of CRISPR-Cas9 System and CRISPR-Cpf1 System in Multigenome Editing

基因编辑技术是指在基因靶位点引入核酸序列变化的一类技术,已广泛应用于生物学、基础医学等多个领域。随着对CRISPR系统研究的不断深入,利用Cas9蛋白进行的多重基因组编辑技术得到了飞速发展,科学家们开发了多种依赖Cas9蛋白的多g RNA载体构建策略,并且在多个物种中已经实现多重基因编辑。而CRISPR-Cpf1系统是基因编辑技术的新工具,极大地丰富了CRISPR/Cas系统库,该系统不仅进一步扩大了基因编辑靶位点的选择范围,同时有效降低了脱靶效应。而且,Cpf1不同于Cas9蛋白的分子作用机制,具有多重编辑的天然优势,已广泛引起人们的关注。该文重点介绍了主要的4种CRISPR-Cas9多重编辑构建策略:Golden Gate Assembly、Multiplexed Lentiviral Expression Cassettes、Polycistronic-t RNA-g RNA Cassettes、Csy4-Cleavable Cassettes和CRISPR-Cpf1多重基因编辑新技术,同时比较了CRISPR-Cas9和CRISPR-Cpf1两种多基因编辑技术的特点,以期为多重基因编辑技术在生物研究领域的应用提供参考。

Gene editing technology is a kind of technology that introduces nucleic acid sequence changes into gene target,which has been widely used in biology,basic medicine and other fields.With the deepening of the research on CRISPR system,the multiple gene editing technology using Cas9 protein has developed rapidly.Scientists have developed a variety of multiple gRNAs vector construction strategies relying on Cas9 protein,which has realized multiple gene editing in various species.CRISPR-Cpf1 system is a new tool of gene editing technology,which has greatly enriched the CRISPR/Cas system library.It not only further expands the selection range of gene editing target sites,and causes less off-target effects,but also its molecular mechanism of action different from Cas9 protein confers it natural advantage in multiple gene editing,which has attracted extensive attention.In this paper,we mainly introduced four construction strategies of multiple gene editing based on CRISPR-Cas9 system,including Golden Gate Assembly,Multiplexed Lentiviral Expression Cassettes,Polycistronic-tRNA-Grna Cassettes,Csy4-cleavable Cassettes,and CRISPR-Cpf1 multi-gene editing technology.Meanwhile,we compare the characteristics of CRISPR-Cas9 system and the CRISPR-Cpf1 system,and expect to provide reference for the application of multiple gene editing technology in the field of biological research.