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

Alyona Sorokina,Alexander Artyuhov,Erdem Dashinimaev,Alexandra Goltsova

doi:10.3389/fmolb.2022.805931

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%.

Highlights

The transmembrane protein CCR5 (CD195) is one of the co-receptor binding sites of human immunodeficiency virus (HIV) (Liu et al, 1996; Venuti et al, 2017)
Because CRISPR manipulation often results in large deletions at the target loci, we could have encountered a situation where, in a putative homozygous mutant clone of MT4-14, one CCR5 allele received the desired 32-nucleotide deletion, while the other allele was so severely disrupted that it would not be detectable by PCR
We measured the gene copy number variation (CNV) by droplet digital polymerase chain reaction (ddPCR) and found that both mutant alleles were present in the MT4-14 line (Supplementary Figure S3.)

Summary

Introduction

The transmembrane protein CCR5 (CD195) is one of the co-receptor binding sites of human immunodeficiency virus (HIV) (Liu et al, 1996; Venuti et al, 2017). There is a mutant form of the CCR5 gene resulting from a deletion of 32 nucleotides in the coding sequence (CCR5Δ32) that causes a frameshift, the appearance of untimely stop codons and knockout of the gene function (Hütter et al, 2011) This mutation is present in approximately 10 and 1% of the population of Northern Europe in heterozygous and homozygous variants, respectively (Stephens et al, 1998; Trecarichi et al, 2006; Muxel et al, 2008). There is a need to create systems for accurate quantification of CCR5Δ32 mutant alleles in heterogeneous cell mixtures that are both efficient and simple enough to translate the method into clinical diagnostic laboratories

Objectives

Methods

Results

Discussion

Conclusion