利用CRISPR/Cas9技术构建斑马鱼sf3a1基因敲除突变体

目的 剪接因子SF3A (splicing factor 3a) 的三个亚基(SF3A1、SF3A2、SF3A3)对U2 snRNP的功能发挥和前剪接小体(prespliceosome)的组装至关重要。SF3A1突变会导致骨髓增生异常综合征(MDS)、慢性粒细胞白血病(CMML)和急性粒细胞白血病(AML)等血液系统疾病。为了研究SF3A1/Sf3a1在生物体中的详细生理功能及其调控机制,本文构建了斑马鱼sf3a1基因敲除突变体。方法 利用CRISPR/ Cas9技术构建斑马鱼sf3a1突变体,通过T7酶切及基因组测序筛选得到F0代及能够稳定遗传的F1代突变体,F1代自交后得到F2代,观察sf3a1纯合缺失对斑马鱼生长发育的影响。结果 基因型鉴定结果显示,本研究得到了2种不同类型的突变品系,分别为-7 bp和-2+13 bp。杂合sf3a1突变体正常存活且可育,而合子型纯合突变体斑马鱼胚胎发育出现明显异常,24 hpf时期胚胎细胞出现大量凋亡,胚胎发育迟缓,并于72 hpf之前死亡。结论 研究利用CRISPR/Cas9技术成功构建了斑马鱼sf3a1基因敲除突变体,为探索sf3a1对斑马鱼生长发育的影响提供了实验材料和依据。

Construction of sf3a1 knockout mutant in zebrafish via CRISPR/Cas9

Objective Splicing factor 3A subunit 1 (SF3A1) is essential for the function of U2 snRNP and the assembly of prespliceosome. Previous studies show that SF3A1 mutations are associated with hematological diseases including myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CMML) and acute myelogenous leukemia (AML). In order to study the detailed physiological functions and molecular regulatory mechanisms of SF3A1/Sf3a1, sf3a1 gene knockout mutant in zebrafish was constructed. Methods We generated sf3a1 knockout mutant using CRISPR/Cas9 system. F0 and F1 mutants were screened by T7 endonuclease and Sanger sequencings. F2 were obtained after F1 generation self-crossing, observe the effects of sf3a1 homozygous deletion on the growth and development of zebrafish. Results Genotypic identification showed that two independent sf3a1 mutation alleles were generated, which were -7 bp and -2+13 bp respectively. The heterozygous sf3a1 mutants were viable and fertile, while the homozygous mutant embryos showed severe abnormality, ubiquitous cell apoptosis, and development retardation. All the homozygous died before 72 hpf. Conclusion In summary, we generated zebrafish sf3a1 knockout mutants according to CRISPR/Cas9 gene editing approach, and revealed that zebrafish sf3a1 homozygous exhibit developmental defects and embryonic lethal.