Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

作者：Shaohui Zheng, Fang Ting Liang, Yu Zhang, Ji‐Feng Fei, Wei Qin, Yanmei Liu · 发表于：Journal of Visualized Experiments · 年份：2023 · DOI：10.3791/64977 · 被引用次数：11 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、RNA modifications and cancer

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.