Zheng Shaohui, Liang Fang, Zhang Yu, Fei Ji-Feng, Qin Wei, Liu Yanmei
Key Laboratory of Brain, Cognition and Education Science, Ministry of Education; Institute for Brain Research and Rehabilitation, Guangdong Key Laboratory of Mental Health and Cognitive Science, South China Normal University.
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.
J Vis Exp. 2023 Feb 17(192). doi: 10.3791/64977.
CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.
CRISPR/Cas9技术提高了斑马鱼在人类遗传疾病建模、疾病发病机制研究和药物筛选方面的价值，但原间隔序列临近基序（PAM）限制是创建由单核苷酸变异（SNV）引起的人类遗传疾病精确动物模型的主要障碍。到目前为止，一些具有广泛PAM兼容性的SpCas9变体已在斑马鱼中显示出效率。优化后的SpRY介导的腺嘌呤碱基编辑器（ABE）zSpRY-ABE8e以及经过合成修饰的gRNA在斑马鱼中的应用，实现了无PAM限制的高效腺嘌呤-鸟嘌呤碱基转换。本文介绍了一种使用zSpRY-ABE8e在斑马鱼中进行无PAM限制的高效腺嘌呤碱基编辑的方案。通过将zSpRY-ABE8e mRNA和经过合成修饰的gRNA混合物注射到斑马鱼胚胎中，构建了一个具有精确突变的斑马鱼疾病模型，该突变模拟了TSR2核糖体成熟因子（tsr2）的致病位点。该方法为建立用于研究疾病机制和治疗方法的精确疾病模型提供了一个有价值的工具。
