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Proc Natl Acad Sci U S A. 2014 Jul 1;111(26):9591-6. doi: 10.1073/pnas.1407473111. Epub 2014 Jun 9.

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

Author information

1
Department of Medicine and Institute for Human Genetics, lin.ye@ucsf.edu yw.kan@ucsf.edu.
2
Department of Medicine and Institute for Human Genetics,Department of Laboratory Medicine, and.
3
Blood Systems Research Institute, San Francisco, CA 94118; and.
4
Department of Medicine and Institute for Human Genetics,Division of Hematology/Oncology, University of California, San Francisco, CA 94143;
5
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322.
6
Department of Laboratory Medicine, and.
7
Department of Medicine and Institute for Human Genetics.
8
Department of Laboratory Medicine, andBlood Systems Research Institute, San Francisco, CA 94118; andThe Liver Center.
9
Division of Hematology/Oncology, University of California, San Francisco, CA 94143;
10
Department of Medicine and Institute for Human Genetics,Department of Laboratory Medicine, and lin.ye@ucsf.edu yw.kan@ucsf.edu.

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 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.

KEYWORDS:

TTAA site; cellular therapy; homologous recombination; off-site target

PMID:
24927590
PMCID:
PMC4084478
DOI:
10.1073/pnas.1407473111
[Indexed for MEDLINE]
Free PMC Article

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