Guide-Specific Loss of Efficiency and Off-Target Reduction With Cas9 Variants

Authors

Liang Zhang
Wei He
Rongjie Fu
Shuyue Wang
Yiwen Chen
Han Xu

Publication Date

10-13-2023

Journal

Nucleic Acids Research

Abstract

High-fidelity clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA-target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against wild-type SpCas9, we found that ∼20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15-18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in the REC3 domain account for the loss of efficiency. We also observed various degrees of sequence-dependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer learning-based computational framework for the prediction of on-target efficiency and off-target effects with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.

Keywords

CRISPR-Cas Systems, Gene Editing, Mutation, RNA, Guide, CRISPR-Cas Systems, CRISPR-Associated Protein 9

DOI

10.1093/nar/gkad702

PMID

37615574

PMCID

PMC10570041

PubMedCentral® Posted Date

8-24-2023

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes