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

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

doi:10.1093/nar/gkaa605

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.

Highlights

The CRISPR-Cas system is a bacterial immune system and it is widely applied to various organisms for target-specific genome editing[1]
This suggests that 2′-OH recognition of theRNA is largely conserved among Cpf1 proteins and that 2′-OH recognition by CRISPR-Cas12a increases from the protospacer adjacent motif (PAM) (TTTN or TTN) distal to the PAM proximal region
This is in line with previous findings in experiments in which mismatches were sequentially introduced into the gRNA and target DNA heteroduplex regions [18]

Summary

Introduction

The CRISPR-Cas system is a bacterial immune system and it is widely applied to various organisms for target-specific genome editing[1]. One of the CRISPR system, CRISPR-Cas RNA-guided endonuclease is widely used to correct or control genes of interest based on its ability to cut double-stranded DNA [1,2,3,4,5]. CRISPR-Cpf is more sensitive to mismatches between the target DNA and the gRNA than CRISPR-Cas is; when a mismatch is introduced into the seed sequence in the protospacer, its cleavage activity is significantly inhibited [19, 20]. We identified a chimeric guide with high accuracy, without on-target cleavage compensation This novel system is advantageous in terms of safety and has application potential for various purposes in vivo [22, 23], and will eventually be useful for gene therapy for diseases caused by genetic defects

Methods

Results

Discussion

Conclusion