Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing

Mol Ther. 2022 Jan 5;30(1):130-144. doi: 10.1016/j.ymthe.2021.10.026. Epub 2021 Nov 2.


Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4+ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4+ T cell and up to 88% of CD34+ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4+ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.

Keywords: Base editing; CCR5; CRISPR/Cas9; CXCR4; HIV; HSC.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Editing* / methods
  • HIV Infections / genetics
  • HIV Infections / metabolism
  • HIV Infections / therapy
  • HIV-1 / physiology
  • Hematopoietic Stem Cells / metabolism
  • Humans
  • Receptors, CCR5* / genetics
  • Receptors, CCR5* / metabolism
  • Receptors, CXCR4* / genetics
  • Receptors, CXCR4* / metabolism
  • T-Lymphocytes / metabolism


  • CCR5 protein, human
  • CXCR4 protein, human
  • Receptors, CCR5
  • Receptors, CXCR4