摘　要：WBP11是WW结构域结合蛋白家族的一员，参与前体mRNA剪接过程，是重要的剪接因子。斑马鱼作为重要的模式生物之一，其基因组中含有人类WBP11的同源基因wbp11。为了研究WBP11在脊椎动物早期发育过程中的作用机制，利用CRISPR/Cas9技术构建了斑马鱼wbp11基因敲除品系。首先，在wbp11基因的第二个外显子上设计基因敲除靶位点，体外转录合成sgRNA，通过显微注射技术对斑马鱼进行基因敲除，得到F0代嵌合体斑马鱼。随后，将嵌合体斑马鱼与野生型斑马鱼杂交，所得到的F1代斑马鱼进行基因型鉴定，筛选出其中的杂合子斑马鱼，并对杂合子斑马鱼的缺失条带进行测序，结果显示其为wbp11基因的2种缺失突变。让同种突变的F1代杂合子斑马鱼自交，对得到的后代F2进行基因型鉴定，筛选获得了纯合子斑马鱼，表明wbp11基因敲除品系构建成功。经显微镜白光成像观察发现，受精后48 h的斑马鱼出现心包水肿、心脏线性化、骨骼弯曲畸形，这可能是由于wbp11基因突变引起剪接机制异常所导致的。此研究为探究WBP11基因在脊椎动物的早期发育中的作用机制开辟了道路。
关键词：斑马鱼；wbp11；CRISPR/Cas9；基因敲除；杂合子
中图分类号：Q341；Q812                     文献标志码：A                  DOI：10.3969/j.issn.1007-7146.2023.03.004

Abstract: As a member of the WW domain binding protein family, WBP11 is an important splicing factor involved in the pre-mRNA splicing process. Zebrafish, one of the notable model organisms, contains orthological homologous gene wbp11 in its genome. In order to study the mechanism of WBP11 in the early development in vertebrates, a knockout strain of wbp11 in zebrafish was constructed using CRISPR/Cas9 technology. Firstly, gene knockout target sites were designed on the second exon of wbp11 gene, and sgRNA was synthesized by transcription in vitro. After microinjection technology, F0 generation chimeric zebrafish with wbp11-knockout was obtained. Then, the F1 generation zebrafish by crossing chimeric zebrafish with wild-type zebrafish were identified by genotype, and heterozygous zebrafish were screened out. The sequencing results for exact bands of heterozygous zebrafish showed that there were two strains with different sequence deletion. Heterozygous zebrafish of the F1 generation of the same strain were self-crossed. The homozygous zebrafish was screened out via genotyping, meaning the wbp11 gene knockout strain was successfully constructed. The zebrafish with 48 hours post fertilization showed pericardial edema, heart linearization and skeletal curvature obversed by microscopic white light imaging. These defections may be resulted from the abnormal splicing mechanism caused by the deletion of wbp11 gene. This study opens the way to explore the mechanism of WBP11 gene in the early development in vertebrates.
Key words: zebrafish; wbp11; CRISPR/Cas9; gene knockout; heterozygote
（Acta Laser Biology Sinica, 2023, 32(3): 217-225）