基于CRISPR/Cas9系统建立新吉富罗非鱼双等位基因敲除技术——以SLC24A5基因为例  

Establishment of Biallelic Knockout Technique in Nile Tilapia(Oreochromis niloticus)Based on CRISPR/Cas9 System:A Case Study of SLC24A5 Gene

作　　者：张佳聪 鲁纪刚 ZHANG Jiacong;LU Jigang(College of Fisheries and Life Sciences,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306

出　　处：《生物技术进展》2024年第3期442-450,共9页Current Biotechnology

基　　金：国家重点研发计划项目(2022YFD2400800);国家自然科学基金重点项目(32130109)。

摘　　要：目前,簇状规则间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白Cas9系统(CRISPR/Cas9)已经成为提高真核生物基因组编辑效率的主要技术。然而,对于像罗非鱼这样繁殖周期较长的物种,CRISPR/Cas9技术由于低纯合效率,在进行大规模遗传筛选研究时面临一定的困难。为了解决这一问题,以罗非鱼为模型,以SLC24A5基因为例,开发了一种高效的CRISPR/Cas9方法,能够在注射的胚胎中以相对稳定的概率直接实现F_(0)代的双等位基因敲除。具体而言,采用两个高效的gRNA进行混合,Cas9蛋白的浓度为800 ng·μL^(-1),Cas9蛋白与gRNA的质量比例为4:1,注射剂量控制在1 nL,即800 pg的Cas9蛋白和200 pg的gRNA。这一敲除技术使得在新吉富罗非鱼(Oreochromis niloticus)的F_(0)代注射胚胎中,能够直接产生表型外显率(Lv.1、Lv.2、Lv.3和Lv.4)为71%的个体,其中显著表型外显率(Lv.1和Lv.2)为17%。这一技术突破为罗非鱼的遗传筛选提供了便利和高效的手段。Currently,the clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein 9(Cas9)system(CRISPR/Cas9)stands out as a primary technology for enhancing genome editing efficiency in eukaryotes.However,for species with longer reproductive cycles,such as the Nile tilapia,the application of CRISPR/Cas9 technology faces challenges due to its low homozygous efficiency,especially in large-scale genetic screening studies.To solve this problem,a highly efficient CRISPR/Cas9 method was developed using SLC24A5 gene as an example in tilapia,which can directly achieve F0 generation biallelic knockout with a relatively stable probability in injected embryos.Specifically,two highly effective guide RNAs(gRNAs)were used for mixing,the concentration of Cas9 protein was 800 ng·μL-1,the mass ratio of Cas9 protein to gRNA was 4∶1,and the injection dose was controlled at 1 nL,that is,800 pg Cas9 protein and 200 pg gRNA.This knockout technique enabled the direct production of individuals with a significant phenotype expressivity(Lv.1,Lv.2,Lv.3,and Lv.4)of 71%in F0 generation embryos of the new GIFT Nile tilapia,with a significantly phenotypic penetrance(Lv.1 and Lv.2)of 17%.This breakthrough technology provided a convenient and efficient means for genetic screening in Nile tilapia.

关 键 词：新吉富罗非鱼 双等位基因敲除 敲除效率 CRISPR/Cas9 SLC24A5 

分 类 号：Q789[生物学—分子生物学] S965.125[农业科学—水产养殖]