Detection of CCR5Δ32 Mutant Alleles in Heterogeneous Cell Mixtures Using Droplet Digital PCR

Abstract

The C-C chemokine receptor type 5 (CCR5 or CD195) is one of the co-receptor binding sites of the human immunodeficiency virus (HIV). Transplantations of hematopoietic stem cells with the CCR5Δ32 knockout mutation could represent an effective tool for the complete cure of HIV; these methods having passed the stage of proof-of-principle. At the same time, using the modern CRISPR/Cas9 genome editing method, we can effectively reproduce the CCR5Δ32 mutation in any wild-type cells. Thus, the task of searching for and accurately quantifying the content of mutant CCR5Δ32 alleles in heterogeneous cell mixtures becomes relevant. In this study, we describe the generation of an artificial CCR5Δ32 mutation using CRISPR/Cas9 followed by multiplex droplet digital polymerase chain reaction (ddPCR) to quantify its content in cell mixtures. The system we have developed allows us to quickly and accurately measure the content of cells with the CCR5Δ32 mutation, down to 0.8%.

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

“…For instance, CCR5 gene-edited primary human CD4 + T cells were found to be resistant to HIV-1 infection in vitro [ 24 , 52 , 53 , 74 ], recapitulating results obtained with other gene editing tools, such as TALENs and zinc finger nucleases [ 75 , 76 ]. Additionally, the CCR5Δ32/Δ32 mutation has been introduced in cell lines and primary human CD4 + T cells via CRISPR [ 51 , 77 ], with no off-target effects detected [ 51 ]. However, while ZFN-mediated CCR5-knockout CD4 + T cells have been shown to persist in vivo [ 76 ], these numbers can decline rapidly.…”

“…Knockout confers in vitro resistance to HIV infection in differentiated macrophages [74] Multi-lineage differentiation in vitro [53] Minimal off-target modifications detected Multi-lineage engraftment potential in animal model [82] Multi-lineage engraftment potential in animal model In vivo resistance to HIV infection [57] Multi-lineage engraftment potential after autologous HSC transplantation Persistence of low frequencies of CCR5 knockout CD4 + T cells [66] iPSCs No off-target modifications detected iPSC-derived monocytes/macrophages resistant to HIV infection [83][84][85] Primary CD4 + T cells Low transduction efficiency with lentiviral vectors [52] Knockout confers in vitro resistance to HIV infection [24,52,53,74] Introduction of ∆32/∆32 mutation No off-target modifications detected [51] Macrophage or T cell cell-lines CRISPR-mediated KO feasible [78,86,87] Introduction of ∆32/∆32 mutation High fidelity screening method [77] This was expanded upon by Xu et al, where also in an in vivo animal model resistance to HIV infection was observed after CCR5-knockout HSC engraft generated via CRISPR/Cas9 [57]. As discussed, autologous CRISPR-edited CCR5 knockout HSCs have also been successfully transplanted in a patient with HIV and acute lymphocytic leukemia [66].…”