• Hanseop Kim, Wi-Jae Lee, Yeounsun Oh, Seung-Hun Kang, Junho K Hur, Hyomin Lee, WooJeung Song, Kyung-Seob Lim, Young-Ho Park, Bong-Seok Song, Yeung Bae Jin, Bong-Hyun Jun, Cheulhee Jung, Dong-Seok Lee, Sun-Uk Kim, and Seung Hwan Lee.
• Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Korea.
• Nucleic Acids Res. 2020 Sep 4; 48 (15): 8601-8616.

AbstractThe CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

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