The plasmids expressing the Cas12a-nucleases used in this study were designed with a promoter (CAG, EF1a Core, CMV, PGK, for the detailed sequences see Additional file 1: DNA sequences) driven enAsCas12a-HF CDS, 3xHA, and a P2A-mcherry reporter, or a promoter (CAG, EF1a Core, CMV, PGK) driven dAsCas12a-HF CDS fusing with VP64-p65-Rta (VPR) activation domain, or a promoter(CAG, EF1a Core, CMV, PGK) driven rat APOBEC1 fusing with dAsCas12a-HF CDS.

In the current study, we analyzed the effects of four commonly used promoters (CAG, EF1a core, CMV, and PGK) on the CRISPR-Cas12a system, including cleaving activity, targeting specificity, multiplex editing efficiency, gene activation level, and base editing ability, which provided the basic information about the impact of promoters for the CRISPR gene-editing toolkit. Therefore, there is a need for considering the level of the Cas-protein in CRISPR-Cas plasmid systems, and a rational design of a CRISPR editor vector can make this technique play a better function and can be applied to the fitness of their unique properties to the intended purpose. In addition, an in vivo therapy purpose application always required a more accurate editing. Reducing the amount of the Cas-protein in the cells improved the targeting specificity but affected the efficiency. Although it had been shown that different promoters had different effects on the expression level of the interesting gene in the plasmid vector, the effects of the promoters on CRISPR gene-editing tools, such as editing activity, targeting specificity, transcriptional activation level, and base editing ability, have not been comprehensively elucidated.

For example, the enhancer/promoter is a critical element in an expression vector, and the selection of the CAG promoter had been reported to significantly increase the expression and the stability of the transgene in mammalian cells. To achieve high transgene expression levels, several investigations had been reported by optimizing cis-acting elements in plasmid systems. Plasmid vector systems contain cis-acting elements, such as enhancers, promoters, polyadenylation signals, and other expression elements, all of which can affect the expression levels of the transgene. The plasmid vector platform is the most commonly used vector for the expression of the CRISPR-Cas tools, such as the adeno-associated virus (AAV) system, the most widely used viral vector for in vivo gene-editing tools’ delivery. However, the successful application of this versatile technology requires the essential expression of the Cas-nuclease protein, which can be generated by a rational synthetic design of the expression cassettes.

The CRISPR-Cas systems have been harnessed as powerful tools for a variety of clinical therapy and basic research, including programmable genome editing, gene activation, live imaging, base editing, and primer editing. 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. 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. 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). 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. 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.

Ecrisper design