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Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide

Authors
Kim, HanseopLee, Wi-jaeOh, YeounsunKang, Seung-HunHur, Junho K.Lee, HyominSong, WooJeungLim, Kyung-SeobPark, Young-HoSong, Bong-SeokJin, YeungJun, Bong-HyunJung, CheulheeLee, Dong-SeokKim, Sun-UkLee, Seung Hwan
Issue Date
4-9월-2020
Publisher
OXFORD UNIV PRESS
Citation
NUCLEIC ACIDS RESEARCH, v.48, no.15, pp.8601 - 8616
Indexed
SCIE
SCOPUS
Journal Title
NUCLEIC ACIDS RESEARCH
Volume
48
Number
15
Start Page
8601
End Page
8616
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/53188
DOI
10.1093/nar/gkaa605
ISSN
0305-1048
Abstract
The 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.
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