SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish

Fang Liang, Yu Zhang, Lin Li, Yexin Yang, Ji-Feng Fei, Yanmei Liu () and Wei Qin ()
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Fang Liang: South China Normal University
Yu Zhang: South China Normal University
Lin Li: South China Normal University
Yexin Yang: South China Normal University
Ji-Feng Fei: Guangdong Academy of Medical Sciences
Yanmei Liu: South China Normal University
Wei Qin: South China Normal University

Nature Communications, 2022, vol. 13, issue 1, 1-10

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-31034-8

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