Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection

作者：Lin Ye, Jiaming Wang, Ashley I. Beyer, Fernando Teque, Thomas J. Cradick, Zhongxia Qi, Judy C. Chang, Gang Bao, Marcus O. Muench, Jingwei Yu, Jay A. Levy, Yuet Wai Kan · 发表于：Proceedings of the National Academy of Sciences · 年份：2014 · DOI：10.1073/pnas.1407473111 · 被引用次数：319 · 研究领域：CRISPR and Genetic Engineering、HIV Research and Treatment、RNA Interference and Gene Delivery

Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.