Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan.
Department of Mammalian Regulatory Networks, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
Sci Rep. 2024 Mar 31;14(1):7615. doi: 10.1038/s41598-024-57551-8.
The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.
CRISPR-Cas 系统在体内基因组编辑是治疗多种疾病的基因治疗的强大工具。我们之前开发了 pCriMGET_9-12a 系统，这是一种体内可切割的供体质粒，可通过 Cas9 和 Cas12a 精确靶向地将外源 DNA 进行靶向敲入。在这里，我们表明，通过靶向质粒的水力传递，pCriMGET_9-12a 系统可应用于成年小鼠肝脏中的体外 DNA 框架内敲入。与不可切割的供体质粒相比，体内可切割的 pCriMGET_9-12a 供体质粒显着提高了 CRISPR-Cas9 和 CRISPR-Cas12a 在成年小鼠肝脏中的敲入效率。该策略还实现了无插入缺失突变的报告基因框架内敲入。因此，使用 pCriMGET_9-12a 系统进行体内基因靶向可能有助于在成年器官中建立更安全、更精确、更通用和更有效的基因治疗方法。
