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

Hanseop Kim; Wi Jae Lee; Yeounsun Oh; Seung Hun Kang; Junho K. Hur; Hyomin Lee; Woo Jeung Song; Kyung Seob Lim; Young Ho Park; Bong Seok Song; Yeung Bae Jin; Bong Hyun Jun; Cheulhee Jung; Dong Seok Lee; Sun Uk Kim; Seung Hwan Lee

doi:10.1093/nar/gkaa605

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

Hanseop Kim, Wi Jae Lee, Yeounsun Oh, Seung Hun Kang, Junho K. Hur, Hyomin Lee, Woo Jeung Song, Kyung Seob Lim, Young Ho Park, Bong Seok Song, Yeung Bae Jin, Bong Hyun Jun, Cheulhee Jung, Dong Seok Lee, Sun Uk Kim, Seung Hwan Lee

Department of Biotechnology

Research output: Contribution to journal › Article › peer-review

25 Citations (Scopus)

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.

Original language	English
Pages (from-to)	8601-8616
Number of pages	16
Journal	Nucleic acids research
Volume	48
Issue number	15
DOIs	https://doi.org/10.1093/nar/gkaa605
Publication status	Published - 2020 Sep 4

ASJC Scopus subject areas

Genetics

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title = "Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide",

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. ",

author = "Hanseop Kim and Lee, {Wi Jae} and Yeounsun Oh and Kang, {Seung Hun} and Hur, {Junho K.} and Hyomin Lee and Song, {Woo Jeung} and Lim, {Kyung Seob} and Park, {Young Ho} and Song, {Bong Seok} and Jin, {Yeung Bae} and Jun, {Bong Hyun} and Cheulhee Jung and Lee, {Dong Seok} and Kim, {Sun Uk} and Lee, {Seung Hwan}",

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AU - Kim, Hanseop

AU - Lee, Wi Jae

AU - Oh, Yeounsun

AU - Kang, Seung Hun

AU - Hur, Junho K.

AU - Lee, Hyomin

AU - Song, Woo Jeung

AU - Lim, Kyung Seob

AU - Park, Young Ho

AU - Song, Bong Seok

AU - Jin, Yeung Bae

AU - Jun, Bong Hyun

AU - Jung, Cheulhee

AU - Lee, Dong Seok

AU - Kim, Sun Uk

AU - Lee, Seung Hwan

PY - 2020/9/4

Y1 - 2020/9/4

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

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