Prime Editing using CRISPR-Cas12a and Circular RNAs in Human Cells
    作者: Ronghong Liang, Zixin He, Kevin Tianmeng Zhao, Haocheng Zhu, Jiacheng Hu, Guanwen Liu, Qiang Gao, Meiyan Liu, Rui Zhang, Jinlong Qiu and Caixia Gao
    刊物名称: Nature Biotechnology
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    Genome editing with prime editors (PEs) based on CRISPR-Cas9 are limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE), and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and are 0.93-fold smaller than corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate precise editing of up to 4 genes simultaneously with the nickase prime editors niCPE and sniCPE.