Base Editing-coupled Survival Screening Enabled High-sensitive Analysis of PAM Compatibility and Finding of the New Possible Off-target

Overview

Journal iScience

Publisher Cell Press

Date 2021 Aug 2

PMID 34337358

Citations 1

Authors

Tianyuan Su

Qi Guo

Yi Zheng

Yizhao Chang

Fei Gu

Xuemei Lu

Qingsheng Qi

Affiliations

Soon will be listed here.

Abstract

Base editing (BE) is a promising genome engineering tool for modifying DNA or RNA and has been widely used in various microorganisms as well as eukaryotic cells. Despite the proximal protospacer adjacent motif (PAM) is critical to the targeting range and off-target effect of BE, there is still lack of a specific approach to analyze the PAM pattern in BE systems. Here, we developed a base editing-coupled survival screening method. Using dCas9 from (SpdCas9) and its variants xdCas9 3.7 and dCas9 NG as example, their PAM patterns in BE systems were extensively characterized using the NNNN PAM library with high sensitivity. In addition to the typical PAM recognition features, we observed more unique PAMs exhibiting BE activity. These PAM patterns will boost the finding of potential off-target editing event arising from non-canonical PAMs and provide the guidelines for PAM usage in the BE system.

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PMID: 36763115 PMC: 9911954. DOI: 10.1007/s00253-023-12418-2.

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