A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

Peng Chen,Hongjian Wang,Lei Yin,Yan Wang,Kai Zhao,Xinghua Zhang,Jun Lei,Jin Zhou,Yibin Wan,Huan Liu,Yiliang Zhang,Yongzheng Li,Zhaoxin Liu

doi:10.1186/s13059-020-01989-2

Peng Chen, Hongjian Wang + Show 11 more

Open Access

https://doi.org/10.1186/s13059-020-01989-2

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Abstract

BackgroundAsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. However, the C-containing PAM (CTTV, TCTV, TTCV, etc.) recognition by Cas12a might induce extra off-target edits at these non-canonical PAM sites.ResultsHere, we identify a novel Cas12a nuclease CeCas12a from Coprococcus eutactus, which is a programmable nuclease with genome-editing efficiencies comparable to AsCas12a and LbCas12a in human cells. Moreover, CeCas12a is revealed to be more stringent for PAM recognition in vitro and in vivo followed by very low off-target editing rates in cells. Notably, CeCas12a renders less off-target edits located at C-containing PAM at multiple sites compared to LbCas12a and AsCas12a, as assessed by targeted sequencing methods.ConclusionsOur study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications.

Highlights

AsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal
Cas12a orthologs with different stringencies for recognizing canonical TTTV and non-canonical Ccontaining PAMs in vitro Cas12a (Cpf1) nucleases such as AsCas12a, LbCas12a, and FnCas12a have been used as gene-editing tools in biological researches with the requirement of recognition of specific PAM sequences, and non-canonical PAM regions were recognized in some extent [14, 15, 20]
With the purpose to check the characters of these Cas12a orthologs for genome editing, we measured dsDNA cleavage activities of all these Cas12a orthologs in vitro using linear dsDNA substrates with 23-nt target sequences and the TTTA PAM (Additional file 1: Figure S8A)

Summary

Introduction

AsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. Besides the most commonly used Streptococcus pyogenes Cas (SpCas9), a series of Cas orthologs from different organisms, such as Staphylococcus aureus (Sa), Streptococcus thermophilus (St), and Neisseria meningitidis (Nm), Recently, CRISPR-Cas12a/Cpf was reported to be a highly specific programmable nuclease with high efficiency comparable to Cas9 [14,15,16]. Several different features make Cas12a an important expansion of CRISPRbased genome-editing tools [14]. Cas12a requires thymine-rich protospacer adjacent motif (PAM) sequence at the 5′ end of the protospacer, different from the guanine-rich PAM sequences at the 3′ end of the target DNA for Cas systems. BV3L6, AsCas12a, and Lachnospiraceae bacterium ND 2006, LbCas12a) [17, 18]

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