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Prime editing using CRISPR-Cas12a and circular RNAs in human cells
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Prime editing using CRISPR-Cas12a and circular RNAs in human cells
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Journal Article
Prime editing using CRISPR-Cas12a and circular RNAs in human cells
2024
Overview
Genome editing with prime editors based on CRISPR-Cas9 is 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 possess a potential multiplexing capacity in comparison to 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 low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.
Prime editing with CRISPR-Cas12a broadens targeting scope and enables multiplexing.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ Bacterial Proteins - genetics
/ Bacterial Proteins - metabolism
/ Biomedical and Life Sciences
/ Biomedical Engineering/Biotechnology
/ CRISPR
/ CRISPR-Associated Proteins - genetics
/ CRISPR-Associated Proteins - metabolism
/ CRISPR-Cas Systems - genetics
/ Editing
/ Endodeoxyribonucleases - genetics
/ Endodeoxyribonucleases - metabolism
/ Humans
/ Nuclease
/ RNA
