Key Laboratory of Brain, Cognition and Education Sciences, Ministry of Education; Institute for Brain Research and Rehabilitation, South China Normal University, 510631, Guangzhou, Guangdong, China.
Institute of Modern Aquaculture Science and Engineering, Guangdong Provincial Engineering Technology Research Center for Environmentally-Friendly Aquaculture, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Sciences, South China Normal University, 510631, Guangzhou, Guangdong, China.
Nat Commun. 2022 Jun 14;13(1):3421. doi: 10.1038/s41467-022-31034-8.
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.
精确的基因修饰在模式生物中对于生物医学研究至关重要。最近无 PAM 碱基编辑器的发展使得评估动物核苷酸突变的功能影响和致病性成为可能。在这里，我们首先通过纯化蛋白与合成修饰的 gRNA 来优化 SpG 和 SpRY 系统在斑马鱼中的作用。SpG 在 NGN PAM 位点具有很高的编辑效率，而 SpRY 则能有效地编辑斑马鱼基因组中的无 PAM 位点。然后，我们生成了 SpRY 介导的胞嘧啶碱基编辑器 SpRY-CBE4max 和 SpRY 介导的腺嘌呤碱基编辑器 zSpRY-ABE8e。两者的目标是放松 PAM，编辑效率高达 96%，产物纯度高。有了这些工具，一些以前无法获得的与疾病相关的遗传变异在斑马鱼中产生，支持了高分辨率靶向在基因组编辑应用中的应用。我们的研究显著提高了 CRISPR-Cas 在斑马鱼基因组景观中的靶向性，促进了该模式生物在揭示基因功能、生理机制和疾病发病机制中的应用。
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