Fusion,of SpCas9 to E-coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells
Fusion,of SpCas9 to E-coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells
复制标题
DOI：
10.1016/j.jbiotec.2017.02.024
复制
发表时间：
2017-04-10
影响因子：
4.1
通讯作者：
Luo, Yonglun
中科院分区：
文献类型：
--
作者：
Lin, Lin;Petersen, Trine Skov;Luo, Yonglun
在工程化的成簇规则间隔短回文重复序列 (CRISPR) 相关蛋白 Cas9 诱导双链 DNA 断裂 (DSB) 后，哺乳动物细胞通过一系列不同途径修复双链 DNA 断裂 (DSB)。虽然 CRISPR-Cas9 因此能够对基因组进行预先设计的修改，但 CRISPR-Cas9 介导的基因组编辑的应用经常受到哺乳动物细胞中不可预测且变化的 DSB 修复途径的阻碍。在这里，我们提出了一种将 Cas9 与重组蛋白融合的策略，用于微调哺乳动物细胞中的 DSB 修复偏好。通过将化脓性链球菌 Cas9 (SpCas9) 与大肠杆菌的重组蛋白 A (Rec A, NP_417179.1) 融合，我们创建了一种重组 Cas9 蛋白 (rSpCas9)，该蛋白可增强哺乳动物细胞中 DSB 位点 indel 突变的产生，通过同源定向单链退火 (SSA) 增加 DSB 修复频率，并抑制同源定向单链退火基因转化约33%。因此，我们的研究首次证明，将 SpCas9 与重组蛋白融合可以影响哺乳动物细胞中 DSB 修复途径之间的平衡。这种方法可能为进一步研究重组 Cas9 蛋白在真核细胞中微调 DSB 修复途径的应用奠定基础。(C) 2017 Elsevier B.V. 保留所有权利。
Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells.(C) 2017 Elsevier B.V. All rights reserved.