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

doi:10.19586/j.2095-2341.2024.0059

生物技术进展 ›› 2024, Vol. 14 ›› Issue (3): 442-450.DOI: 10.19586/j.2095-2341.2024.0059

• 研究论文 • 上一篇

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

张佳聪(), 鲁纪刚()

上海海洋大学水产与生命学院，上海 201306

收稿日期:2024-03-25 接受日期:2024-04-18 出版日期:2024-05-25 发布日期:2024-06-18
通讯作者: 鲁纪刚
作者简介:张佳聪E-mail：1659346822@qq.com；
基金资助:
国家重点研发计划项目(2022YFD2400800);国家自然科学基金重点项目(32130109)

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

Jiacong ZHANG(), Jigang LU()

College of Fisheries and Life Sciences，Shanghai Ocean University，Shanghai 201306，China

Received:2024-03-25 Accepted:2024-04-18 Online:2024-05-25 Published:2024-06-18
Contact: Jigang LU

摘要/Abstract

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F₀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F₀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。

关键词: 新吉富罗非鱼, 双等位基因敲除, 敲除效率, CRISPR/Cas9, SLC24A5

Abstract:

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.

Key words: New Gift Nile tilapia, biallelic knockout, knockout efficiency, CRISPR/Cas9, SLC24A5

中图分类号:

Q789

张佳聪, 鲁纪刚. 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[J]. 生物技术进展, 2024, 14(3): 442-450.

Jiacong ZHANG, Jigang LU. Establishment of Biallelic Knockout Technique in Nile Tilapia (Oreochromis niloticus) Based on CRISPR/Cas9 System: A Case Study of SLC24A5 Gene[J]. Current Biotechnology, 2024, 14(3): 442-450.

图/表 12

图1 靶点设计示意图

Fig. 1 Target design schematic

表1 本研究所用序列

Table 1 The sequence used in this study

名称	序列（5'→3'）	用途
crRNA1	5'-GATCACTAATACGACTCACTATAGGAAACTCGGAGGACTGTGGGTTTTAGAGCTAGAAATAGC-3'	合成gRNA
crRNA2	5'-GATCACTAATACGACTCACTATAGGATTGTCACAGGACGTAGCGTTTTAGAGCTAGAAATAGC-3'	合成gRNA
crRNA3	5'-GATCACTAATACGACTCACTATAGGTCAGCACCATTGTGGGATGTTTTAGAGCTAGAAATAGC-3'	合成gRNA
tracrRNA	5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'	合成gRNA
SLC24A5-F	5'-GACTGACTAAATAAGCCCACAGT-3'	扩增SLC24A5
SLC24A5-R	5'-AGACGCCCAGCCTGATAAAG-3'

表2 纯化后DNA浓度

Table 2 DNA concentration after purification

名称	浓度/(ng·μL^-1)
SLC24A5-1	301.5
SLC24A5-2	289.9
SLC24A5-3	296.3

表3 纯化后RNA浓度

Table 3 RNA concentration after purification

RNA	浓度/(ng·μL^-1)
SLC24A5-1	598.3
SLC24A5-2	600.9
SLC24A5-3	609.2

图2 罗非鱼F0敲除策略示意图

Fig. 2 Diagram of the F0 knockout approach in tilapia

表4 Cas9蛋白与gRNA配比表 ( μL)

Table 4 Ratio of Cas9 protein to gRNA

组名	SLC24A5-1	SLC24A5-2	SLC24A5-3	Cas9蛋白	DEPC水
Expt1	0	1.67	0	1	2.33
Expt2	0	0.82	0.83	1	2.35
Expt3	0.56	0.56	0.55	1	2.33

图3 本实验中出现的眼部色素Lv.1~Lv.5表型A：眼部色素面积占比为0%~5%的幼鱼；B：眼部色素面积占比为6%~20%的幼鱼；C:眼部色素面积占比为21%~50%的幼鱼；D：眼部色素面积占比为51%~90%的幼鱼；E：眼部色素面积占比为91%~100%的幼鱼，A、B、C、D、E的左图为全身图，右图为眼部放大图，白色虚线圈出了眼部色素变化区域。

Fig. 3 The phenotype of eye pigment Lv.1~Lv.5 appeared in this experiment

图4 SLC24A5不同单靶点注射后的新吉富罗非鱼存活率与色素缺失率A：存活率；B：色素缺失率。本实验进行3次重复。NS.—无统计学差异；*，**，***分别表示2组数据间在P<0.05,P<0.01,P<0.001水平上有统计学意义的差异。

Fig. 4 Survival rate and pigmentation loss rate of the New Gift Nile Tilapia after SLC24A5 injection at different single targets

图5 SLC24A5不同数量的靶点混合后注射的新吉富罗非鱼存活率与色素缺失率注：A：存活率；B：色素缺失率。本实验进行3次重复。Expt1—单靶点；Expt2—双靶点；Expt3—三靶点；NS.—无统计学差异；*，**，***分别表示2组数据间差异在P<0.05,P<0.01,P<0.001水平上有统计学意义。

Fig. 5 Survival rate and pigment loss rate of New Gift Nile tilapia after injection with different quantities of SLC24A5 targets

图6 不同靶点数量注射的罗非鱼的表型外显率

Fig. 6 Phenotypic penetrance of difterent quantities of targets in tilapia

图7 未注射组和注射组SLC24A5 PCR产物琼脂糖电泳图

Fig. 7 The agarose gel electrophoresis images of SLC24A5 PCR products for the non-injected and injected groups

图8 每条鱼的变异读数百分比和具有移码突变的读数百分比Sanger测序结果

Fig. 8 Percentage of reads mutated and reads with a frameshift mutation for each fish from Sanger sequencing results

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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

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