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High-resolution Inference of Multiplexed Anti-HIV Gene Editing using Single-Cell Targeted DNA Sequencing

Mohamed S Bouzidi, Zain Y Dossani, Carolina Di Benedetto, Kyle A Raymond, Shivani Desai, Leonard R Chavez, Paola Betancur, Satish K Pillai
bioRxiv Jan 2024

Gene therapy-based HIV cure strategies typically aim to excise the HIV provirus directly, or target host dependency factors (HDFs) that support viral persistence. Cure approaches will likely require simultaneous co-targeting of multiple sites within the HIV genome to prevent evolution of resistance, and/or co-targeting of multiple HDFs to fully render host cells refractory to HIV infection. Bulk cell-based methods do not enable inference of co-editing within individual viral or target cell genomes, and do not discriminate between monoallelic and biallelic gene disruption. Here, we describe a targeted single-cell DNA sequencing (scDNA-seq) platform characterizing the near full-length HIV genome and 50 established HDF genes, designed to evaluate anti-HIV gene therapy strategies. We implemented the platform to investigate the capacity of multiplexed CRISPR-Cas9 ribonucleoprotein complexes (Cas9-RNPs) to simultaneously 1) inactivate the HIV provirus, and 2) knockout the CCR5 and CXCR4 HDF (entry co-receptor) genes in microglia and primary monocyte-derived macrophages (MDMs). Our scDNA-seq pipeline revealed that antiviral gene editing is rarely observed at multiple loci (or both alleles of a locus) within an individual cell, and editing probabilities across sites are linked. Our results demonstrate that single-cell sequencing is critical to evaluate the true efficacy and therapeutic potential of HIV gene therapy.



Viral Latency

Institution Type


Indication / Modality

HIV Viral Latency

Goal of Study

Genome Editing

PAD Project


Analytes Assessed


Proof Point Demonstrated

Better than Bulk, Zygosity