高保真CRISPR/Cas9系统在家禽细胞中的编辑特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0673

摘要/Abstract

摘要：

【目的】高保真编辑器有望大范围降低传统CRISPR/Cas9系统在家禽基因编辑过程中发生的脱靶效应（off-target），但是其在家禽的应用目前仍然缺乏数据支持。为了评估高保真编辑器在家禽细胞中靶向（on-target）的编辑效果与脱靶特性，从而对家禽的分子遗传育种和种质资源利用提供有效参考。【方法】选取5种目前已被报道且具有较高精确性的高保真Cas9变体，分别与设计的sgRNA共同对鸡的DF-1细胞进行转染，收集阳性细胞进行基因测序，并对不同的高保真编辑器进行编辑效率与脱靶效应的平行对比。【结果】在针对禽流感抗性相关基因ANP32B和生殖相关基因DAZL的多个sgRNA测试中，eCas9维持较高的编辑活性的同时表现出了较低的脱靶效应。同时，在检测的多种高保真变体中，SuperFiCas9具有最低脱靶效应，但是在家禽DF-1细胞的基因编辑中会发生明显的编辑效率下降。【结论】多种高保真编辑器在家禽细胞内均展现编辑活性，其中eCas9编辑器具有高效、低脱靶的特性，可作为鸡重要经济性状的遗传改良和分子育种工作的优选高保真编辑工具。

关键词: 鸡, 高保真编辑器, 靶向, 脱靶, ANP32B

Abstract:

【Objective】 The emergence of high-fidelity editors in recent years is expected to widely reduce the off-target effect that occurs during gene editing in poultry with the conventional CRISPR/Cas9 system. However, there is still a lack of data support for the application of these high-fidelity editors in poultry. This study aims to further evaluate their on-target editing effects and off-target characteristics in poultry cells, so as to provide an effective reference for molecular genetic breeding and germplasm resource utilization in poultry.【Method】 Five high-fidelity Cas9 variants, which have been reported with high accuracy, were selected and co-transfected with the designed sgRNAs in chicken DF-1 cells, and the positive cells were collected for gene sequencing, and the editing efficiency and off-target effect of different high-fidelity editors were compared in parallel.【Result】 eCas9 maintained high editing activity while demonstrating low off-target effects in multiple sgRNAs tested against the avian influenza resistance-related gene ANP32B and the reproduction-related gene DAZL. Meanwhile, SuperFiCas9 had the lowest off-target effect among multiple tested high-fidelity variants, but a significant decrease in editing efficiency occurred in gene editing of poultry DF-1 cells.【Conclusion】 A variety of high-fidelity editors have shown editing activity in poultry cells, among which the eCas9 editor has high efficiency and low off-target properties, and can be used as a preferred high-fidelity editing tool for the genetic improvement of economically important traits and molecular breeding work in chickens.

Key words: chicken, high-fidelity editor, on-target, off-target, ANP32B

焦丹荣, 马梦雪, 何柏水, 谢龙, 左二伟. 高保真CRISPR/Cas9系统在家禽细胞中的编辑特性研究[J]. 生物技术通报, 2024, 40(10): 191-197.

JIAO Dan-rong, MA Meng-xue, HE Bai-shui, XIE Long, ZUO Er-wei. Editing Features of High-fidelity CRISPR/Cas9 System in Poultry Cells[J]. Biotechnology Bulletin, 2024, 40(10): 191-197.

图/表 4

表1 引物列表

Table 1 Primer list

引物名称Primer name	sgRNA打靶位点sgRNA target-sites（5'-3'）	引物序列Primer sequence（5'-3'）	基因Gene
SgRNA1	ATAACTGCCGTTCAGATGAT	F：ATAACTGCCGTTCAGATGAT R：ATCATCTGAACGGCAGTTATC	ANP32B
SgRNA2	GGAGATTGGAGATGGACAGC	F：GAGATTGGAGATGGACAGC R：GCTGTCCATCTCCAATCTCC	ANP32B
SgRNA3	GTCCATCCCCATCTGCCTCA	F：TCCATCCCCATCTGCCTCA R：TGAGGCAGATGGGGATGGAC	ANP32B
SgRNA4	GGAAGAGGAGGATGATGATG	F：GAAGAGGAGGATGATGATG R：CATCATCATCCTCCTCTTCC	ANP32B
SgRNA5	TGATCTGGAAGGTGAAGAGG	F：TGATCTGGAAGGTGAAGAGG R：CCTCTTCACCTTCCAGATCAC	ANP32B
SgRNA6	TGAACAATATGGTACTGTGA	F：TGAACAATATGGTACTGTGA R：TCACAGTACCATATTGTTCAC	DAZL

图1 sgRNA质粒的构建流程以及不同高保真编辑器的图谱 a：sgRNA质粒的构建流程；b：编辑器质粒结构图谱

Fig. 1 Construction process of plasmid sgRNA and mapping of different high-fidelity editors a: Construction process of sgRNA plasmid; b: the structure of plasmid editors

图2 不同高保真编辑器在鸡的ANP32B、DAZL基因上的编辑特性 a：高保真编辑器在鸡细胞编辑特性研究的实验流程；b：不同高保真编辑器在ANP32B、DAZL基因多个位点的编辑效率对比；c：不同高保真编辑器在ANP32B-site5靶位点中具体编辑情况；d：不同编辑器在ANP32B-site5靶位点的编辑窗口

Fig. 2 Editing features of different high-fidelity editors in chicken gene ANP32B and DAZL a: The experiment schedule of gene editing in chicken cells for high-fidelity editors; b: editing efficiency comparing of different high-fidelity editors in different target sites from ANP32B and DAZL; c: detailed editing characteristics of different high-fidelity editors in ANP32B-site5 target site; d: the editing windows of different high-fidelity editors in ANP32B-site5

图3 不同高保真编辑器在鸡的ANP32B、DAZL基因编辑时的脱靶情况 a：不同高保真编辑器在鸡的ANP32B-site1、3、4位点的脱靶情况；b：不同高保真编辑器在ANP32B-site4-off7位点上发生的具体脱靶情况；c：不同高保真编辑器的on-target与off-target的联合分析

Fig. 3 Off-target editing of different high-fidelity editors in editing chicken's ANP32B and DAZL loci a: The off-target effects at ANP32B-site1, 3, 4 of different Cas9 variants; b: the detail off-target characteristics induced by different Cas9 varaints at ANP32B-site4-off7 locus; c: joint analysis of the on-target and off-target effects of Cas9 variants

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