Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5 Delta 32 mutation confers resistance to HIV infection

• Type of Material: Article
• Authors: Lin Ye, University of California San Francisco; Jiaming Wang, University of California San Francisco; Ashley I. Beyer, Blood Systems Research Institute; Fernando Teque, University of California San Francisco; Thomas J. Cradick, Georgia Institute of Technology; Zhongxia Qi, University of California San Francisco; Judy C. Chang, University of California San Francisco; Gang Bao, Emory University; Marcus O. Muench, University of California San Francisco; Jingwei Yu, University of California San Francisco; Jay A. Levy, University of California San Francisco; Yuet Wai Kan, University of California San Francisco
• Language: English
• Date: 2014-07-01
• Publisher: NATL ACAD SCIENCES
• Publication Version: Final Published Version
• Copyright Statement: © 2014 National Academy of Sciences
• Final Published Version (URL): http://dx.doi.org/10.1073/pnas.1407473111
• Title of Journal or Parent Work: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• Volume: 111
• Issue: 26
• Start Page: 9591
• End Page: 9596
• Grant/Funding Information: This work was supported by National Institutes of Health Grants AI 102825 and P01DK088760 and the UCSF Helmut Horten fund.
• Keywords: KNOCKDOWN; RNA; MACROPHAGES; homologous recombination; TTAA site; DEFINED FACTORS; Multidisciplinary Sciences; ZINC-FINGER NUCLEASES; off-site target; GENE CORRECTION; Science & Technology - Other Topics; Science & Technology; CAS9; SPECIFICITY; cellular therapy; CCR5; TRANSDUCTION
• Research Categories: Biology, Cell; Engineering, Biomedical

Abstract

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 RNAguided 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.