高保真CRISPR/Cas9系统在家禽细胞中的编辑特性研究

Editing Features of High-fidelity CRISPR/Cas9 System in Poultry Cells

【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。

【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 highfidelity 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.