Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.
Funder
the National Key R&D Program of China
the National Natural Science Foundation of China
the Fundamental Research Funds for the Central Universities
the China Postdoctoral Science Foundation
Publisher
Public Library of Science (PLoS)
Cited by
1 articles.
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