当前位置: X-MOL 学术Enzyme Microb. Technol. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2020-08-01 , DOI: 10.1016/j.enzmictec.2020.109556
Yankun Yang 1 , Guoqiang Liu 1 , Xiao Chen 1 , Meng Liu 1 , Chunjun Zhan 1 , Xiuxia Liu 1 , Zhonghu Bai 1
Affiliation  

Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.

中文翻译:

高效 CRISPR/Cas9 基因组编辑系统,在毕赤酵母中具有可消除的游离 sgRNA 质粒

Pichia pastoris 是一种甲基营养型酵母,由于强诱导型酒精氧化酶启动子 (PAOX1),在其中开发了蛋白质的宿主异源表达。然而,很难在 P. pastoris 中操纵基因组。基于之前将 CRISPR/Cas9 系统应用于毕赤酵母的尝试,开发了带有附加型 sgRNA 质粒的 CRISPR/Cas9 系统,获得了 100% 的基因组编辑效率、高多拷贝基因编辑和稳定的多基因编辑,而没有因多 sgRNA。并且 28/34 (∼82 %) sgRNAs 测试是有效的。CGG 可能具有比其他三个 NGG 基序略高且更稳定的切割效率,并且低 GC 含量可能更适合更高的切割效率。这为研究人员提供了一个稳定的基因组编辑工具,显示出高编辑效率,缩短实验周期。此外,我们将 dCas9 引入 P. pastoris 并实现了目标基因干扰,扩展了 P. pastoris 中的 CRISPR/Cas9 工具箱。
更新日期:2020-08-01
down
wechat
bug