SpG and SpRY Variants Expand the CRISPR Toolbox for Genome Editing in Zebrafish

Overview

Journal Nat Commun

Specialty Biology

Date 2022 Jun 14

PMID 35701400

Authors

Fang Liang

Yu Zhang

Lin Li

Yexin Yang

Ji-Feng Fei

Yanmei Liu

Wei Qin

Affiliations

Soon will be listed here.

Abstract

Precise genetic modifications in model organisms are essential for biomedical research. The recent development of PAM-less base editors makes it possible to assess the functional impact and pathogenicity of nucleotide mutations in animals. Here we first optimize SpG and SpRY systems in zebrafish by purifying protein combined with synthetically modified gRNA. SpG shows high editing efficiency at NGN PAM sites, whereas SpRY efficiently edit PAM-less sites in the zebrafish genome. Then, we generate the SpRY-mediated cytosine base editor SpRY-CBE4max and SpRY-mediated adenine base editor zSpRY-ABE8e. Both target relaxed PAM with up to 96% editing efficiency and high product purity. With these tools, some previously inaccessible disease-relevant genetic variants are generated in zebrafish, supporting the utility of high-resolution targeting across genome-editing applications. Our study significantly improves CRISPR-Cas targeting in the genomic landscape of zebrafish, promoting the application of this model organism in revealing gene function, physiological mechanisms, and disease pathogenesis.

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