当前位置: X-MOL 学术3 Biotech › 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.)
Improved efficiency of genome editing by constitutive expression of Cas9 endonuclease in genetically-modified mice
3 Biotech ( IF 2.6 ) Pub Date : 2021-01-11 , DOI: 10.1007/s13205-020-02580-z
Bita Ghassemi 1 , Monire Jamalkhah 2 , Gelareh Shokri 3 , Mousa Kehtari 4 , Masoud Soleimani 5 , Mehdi Shamsara 6 , Jafar Kiani 7, 8
Affiliation  

Despite its convenience and precision, CRISPR-based gene editing approaches still suffer from off-target effects and low efficiencies, which are partially rooted in Cas9, the nuclease component of the CRISPR/Cas9 system. In this study, we showed how mouse genome editing efficiency can be improved by constitutive and inheritable expression of Cas9 nuclease. For this goal, a transgenic mouse line expressing the Cas9 protein (Cas9-mouse) was generated. For in vitro assessment of gene editing efficiency, the Cas9-mice were crossed with the EGFP-mice to obtain mouse embryonic fibroblasts (MEF) expressing both EGFP and Cas9 (MEFCas9-EGFP). Transfection of these cells with in vitro transcribed (IVT) EGFP sgRNA or phU6-EGFPsgRNA plasmid led to robust decrease of Mean Fluorescent Intensity (MFI) to 8500 ± 1025 a.u. and 13,200 ± 1006 a.u. respectively. However, in the control group, in which the MEFEGFP cells were transfected with a pX330-EGFPsgRNA plasmid, the measured MFI was 16,800 ± 2254 a.u. For in vivo assessment, the Cas9-zygotes at two pronuclei stage (2PN) were microinjected with a phU6-HhexsgRNA vector and the gene mutation efficiency was compared with the wild-type (WT) zygotes microinjected with a pX330-HhexsgRNA plasmid. The analysis of born mice showed that while the injection of Cas9-zygotes resulted in 43.75% Hhex gene mutated mice, it was just 15.79% for the WT zygotes. In conclusion, the inheritable and constitutive expression of Cas9 in mice provides an efficient platform for gene editing, which can facilitate the production of genetically-modified cells and animals.



中文翻译:

通过在转基因小鼠中组成性表达 Cas9 核酸内切酶来提高基因组编辑的效率

尽管方便和精确,但基于 CRISPR 的基因编辑方法仍然存在脱靶效应和低效率的问题,这部分源于 CRISPR/Cas9 系统的核酸酶成分 Cas9。在这项研究中,我们展示了如何通过 Cas9 核酸酶的组成型和可遗传表达来提高小鼠基因组编辑效率。为此,产生了表达 Cas9 蛋白的转基因小鼠系 (Cas9-mouse)。对于基因编辑效率的体外评估,Cas9 小鼠与 EGFP 小鼠杂交以获得表达 EGFP 和 Cas9 的小鼠胚胎成纤维细胞 (MEF) (MEF Cas9-EGFP )。用体外转录 (IVT) EGFP sgRNA 或 phU6-EGFP sgRNA转染这些细胞质粒导致平均荧光强度 (MFI) 分别大幅下降至 8500 ± 1025 au 和 13,200 ± 1006 au。然而,在用 pX330-EGFP sgRNA质粒转染 MEF EGFP细胞的对照组中,测量的 MFI 为 16,800 ± 2254 au。对于体内评估,在两个原核阶段 (2PN) 的 Cas9 合子被显微注射将 phU6-Hhex sgRNA载体和基因突变效率与显微注射 pX330-Hhex sgRNA的野生型 (WT) 合子进行比较质粒。对出生小鼠的分析表明,虽然注射 Cas9 受精卵导致 43.75% 的 Hhex 基因突变小鼠,但对于 WT 受精卵,这一比例仅为 15.79%。总之,Cas9在小鼠中的可遗传和组成型表达为基因编辑提供了一个有效的平台,可以促进转基因细胞和动物的生产。

更新日期:2021-01-12
down
wechat
bug