质粒载体平台是通用CRISPR-Cas技术表达的最常用载体,启动子是表达载体的关键元件,因此,分析启动子对CRISPR编辑器的影响为基因编辑工具包提供了基本信息,并可作为其设计指南.在这里,我们对四种常用的启动子(CAG,~1700bp;EF1a核心,~210bp;CMV,~500bp;和PGK,〜500bp)在哺乳动物细胞中的CRISPR-Cas12a系统中探索启动子对这一强大工具的影响。我们发现在没有严重损害靶向特异性的情况下,CAG启动子驱动Cas12a编辑器表现出最活跃(效率为100%,特异性指数=~75%)在基因组切割中,多路编辑,转录激活,和基础编辑,其次是启动子CMV(效率=70~90%(vsCAG),特异性指数=~78%),然后是EF1a核心和PGK(效率=40-60%,与CAG相比),但具有更高的特异性(特异性指数=~84%和~82%,分别)。因此,CAG在CRISPR-Cas12a系统中推荐用于需要强大编辑活动但没有大小限制的应用程序。当需要较小的空间时,CMV通常可以替代CAG,EF1a与PGK相似,具有相对较高的特异性,但尺寸较小,因此更适合于体内治疗应用。数据概述了CRISPR-Cas12a系统中广泛使用的启动子的特性,这可以是其应用的指南,也可以是基因编辑领域的有用资源。
The plasmid vector platform is the most commonly used vector for the expression of the versatile CRISPR-Cas technique and the promoter is a crucial element for the expression vector, thus profiling the impact of the
promoters on CRISPR editors provides the basic information for the gene-editing toolkits and can be a guideline for its design. Herein, we made a parallel comparison among four commonly used
promoters (CAG, ~ 1700 bp; EF1a core, ~ 210 bp; CMV, ~ 500 bp; and PGK, ~ 500 bp) in CRISPR-Cas12a system in mammalian cells to explore the impact of
promoters on this powerful tool. We found that without badly damaging targeting specificity, the CAG promoter-driving Cas12a editor exhibited the most active (efficiency takes as 100%, specificity index = ~ 75%) in genomic cleavage, multiplex editing, transcriptional activation, and base editing, followed by promoter CMV (efficiency = 70 ~ 90% (vs CAG), specificity index = ~ 78%), and then EF1a core and PGK (both efficiency = 40-60%, vs CAG) but with higher specificity (specificity index = ~ 84% and ~ 82%, respectively). Therefore, CAG is recommended in the CRISPR-Cas12a system for the applications that need a robust editing activity but without size limitation, CMV mostly can be an alternative for CAG when requiring a smaller space, EF1a is similar to PGK with relatively high specificity, but has a smaller size, thus is more suitable for in vivo therapeutic applications. The data outlined the properties of the widely used
promoters in the CRISPR-Cas12a system, which can be a guide for its applications and can be a useful resource for the gene-editing field.