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

TLDR

Homozygous CCR5Δ32 individuals are naturally resistant to HIV‑1 infection. The study aims to generate CCR5Δ32‑homozygous iPSCs via genome editing to enable a potential functional cure for HIV‑1. Genome editing of wild‑type iPSCs was performed using TALENs or CRISPR‑Cas9 combined with piggyBac transposon technology. Editing yielded high allele targeting, with 14 % biallelic TALEN and 33 % CRISPR efficiency; piggyBac excision produced clean CCR5Δ32, and differentiated cells resisted HIV‑1 infection.

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.