Zhu Xiang-Xing, Pan Jia-Sheng, Lin Tao, Yang Ye-Cheng, Huang Qiu-Yan, Yang Shuai-Peng, Qu Zi-Xiao, Lin Zi-Sheng, Wen Jian-Cong, Yan Ai-Fen, Feng Juan, Liu Lian, Zhang Xiao-Li, Lu Jia-Hong, Tang Dong-Sheng
Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, Gene Editing Technology Center of Guangdong Province, Foshan University, Foshan, 528225, China.
School of Life Sciences and Food Engineering, Hebei University of Engineering, Handan, 056038, China.
Biotechnol Lett. 2022 Jan;44(1):59-76. doi: 10.1007/s10529-021-03214-x. Epub 2022 Jan 8.
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.
基因敲除猪在农业和医学领域具有重要应用。与CRISPR/Cas9相比，腺嘌呤碱基编辑器（ABE）可在基因组中将单个A·T碱基对转换为G·C碱基对，而不会产生DNA双链断裂，并且该方法在猪基因改造中具有更高的准确性和生物安全性。然而，ABE在猪基因敲除中的应用受到原间隔序列相邻基序序列和碱基编辑窗口的限制。可变mRNA剪接是真核生物中形成具有多种功能蛋白质的重要机制。剪接体识别前体mRNA中剪接供体和受体的保守序列。这些保守序列中的突变会导致外显子跳跃，从而产生具有新功能的蛋白质或由于移码突变导致基因失活。在本研究中，利用腺嘌呤碱基编辑介导的外显子跳跃来扩展ABE在基因敲除猪生成中的应用。我们首先构建了一种适用于猪体细胞转染的改良“一体化”ABE载体，其包含用于单碱基编辑的ABE和一个sgRNA表达盒。在对多个内源基因位点进行单碱基编辑期间，“一体化”ABE载体在猪细胞中诱导了高效的sgRNA依赖性A到G的转换。随后，设计了一种ABE系统，用于对猪生长激素受体（GHR）基因中保守的剪接受体位点（内含子5 3'端的AG序列）和剪接供体位点（内含子6 5'端的GT序列）进行单个腺嘌呤编辑；该方法在细胞水平上实现了高效的A到G转换。然后，生成了在GHR内含子5的剪接受体位点携带双等位基因A到G转换的猪单细胞克隆。逆转录-聚合酶链反应（RT-PCR）表明在mRNA水平上外显子6跳跃。蛋白质免疫印迹法显示GHR蛋白缺失，并且基因测序表明不存在sgRNA依赖性脱靶效应。这些结果证明了在猪细胞中精确的腺嘌呤碱基编辑介导的外显子跳跃和基因敲除。这是腺嘌呤碱基编辑介导的外显子跳跃用于猪基因调控的首个概念验证研究，并且这项工作为猪在农业和医学应用中的精确和安全基因改造提供了一种新策略。
