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bioRxiv · 10.1101/2023.02.06.527393

Profiling the impact of the promoters on CRISPR-Cas12a system in human cells

Li, J.·Liang, Q.·Zhou, H.·Zhou, M.·Huang, H.
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Abstract

The plasmid vector platform is the most commonly used 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,[~]1700bp; EF1a core, [~]210bp; CMV, [~]500bp; and PGK, [~]500bp) 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.

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Li, J., Liang, Q., Zhou, H., Zhou, M., Huang, H.. 2023-02-07. Profiling the impact of the promoters on CRISPR-Cas12a system in human cells. https://doi.org/10.1101/2023.02.06.527393

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