CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus

Abstract

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

CRISPRs encounter Cocktail: a dawn for curing HIV/AIDS

“…All the modified iPSCs could differentiate into monocytes/macrophages and gain resistance to HIV-1 challenge [21]. Dual gRNAs resulted in doubled efficiency from 12.5% to 27% of cell colonies and 22.2% to 41% biallelic editing [22]. Delivery systems were recently discussed about in a comprehensive study on primary CD4+ T cell manipulation [23].…”

Redesigning Nature:To Be or not to Be?

“…Kang et al 9 reported that viral entry into immune cells can be blocked by the successful replacement of CCR5 gene in human stem cells using CRISPR/Cas9. Wang et al 10 reported that repairing the DNA fragmented by Cas9 by the error-prone repair machinery of the cell led to mutated sequences that disrupted the viral life cycle.…”

Cell and gene therapy strategies to eradicate HIV reservoirs

“…Induced pluripotent stem cells (iPSCs) are under investigation as well, with the idea that modified iPSCs could differentiate into hematopoietic cells. Ye et al and Kang et al both demonstrated efficient bilallelic disruption of CCR5 in iPSCs and additionally showed efficient differentiation of modified cells [25,26]. Along the same lines, mesenchymal stem cells have been gene edited and converted to CD34+ progenitor cells [27].…”