Table 2.
CRISPR-based methods to edit CCR5.
| Gene editing tool | Approach | En gra ftm ent type | Target cells | Study outcome |
|---|---|---|---|---|
| CRISPR-Cas9 knockout | Reproduced Δ32 mutation | in vitro | Human CD4+T cells | R5-tropic resistance was conferred to primary cells [146]. |
| CRISPR-Cas9 TALENs | CRISPR and TALENs used to reproduce Δ32 mutation | in vitro | iPSCs | CRISPR showed better targeting efficiency than TALENs [115]. |
| CRISPR-Cas9 knockout | Reproduced Δ32 mutation | in vitro | iPSCs, human CD4+T cells | Multiple sgRNA pairs generated CCR5 knockouts and resistance to R5-tropic HIV 116,147]. |
| CRISPR-Cas9 knockout | Reproduced Δ32 mutation | in vitro | TZM-bl cells | Cas9-sgRNA-edited cells showed few off-target events, and lentiviral delivery was safe [148]. |
| CRISPR-Cas9 RNP technology | Reproduced Δ32 mutation | in vitro | Human CD4+T cells | RNP-mediated delivery was safe, disrupted CCR5, and provided R5-tropic resistance [149]. |
| CRISPR-Cas9 knockout | Reproduced Δ32 mutation | in vitro | A549, HeLa, primary human skeletal myoblasts | Δ32 mutation induced resistance to HIV [150]. |
| CRISPR-Cas9 HDR | Reproduced Δ32 mutation | humice | HSPCs | Δ32 HSPCs conferred HIV resistance [134]. |
| CRISPR-Cas9 knockout | Flanking regions of the Δ32 locus were targeted to minimize off-target events | in vitro | Human CD4+T cells, Jurkat Cells | Knockout conferred resistance to R5-tropic virus without off-target effects [151]. |
| CRISPR-Cas9-tracrRNA structure modification | sgRNA structure was investigated to improve indel outcomes for safer CCR5 ablation. | in vitro | Human CD4+T cells | Modified tracrRNA enhanced CCR5 cleavage and improved indel frequency [123]. |
| CRISPR-SaCas9 | S. aureus(Sa) and S. pyogenes(Sp) Cas9s were compared for CCR5 ablating efficiency | humice | CD4+T cells, HSPCs | SaCas9 showed superior CCR5 cleavage efficiency than SpCas9 [152]. |
| CRISPR-Cas9 NHEJ | Series of sgRNAs were assessed for CCR5 ablating efficiency | in vitro | HSCs | CCR5 ablation conferred HIV resistance [153]. |
| CRISPR-Cas9 HDR | Reproduced Δ32 mutation | NH P | HSCs | Autologous CCR5-deficient HSCs provided short-term anti-SIV protection [154]. |
| TALEN and CRISPR-Cas9 | PiggyBac-mediated CRISPR to reproduce the Δ32 mutation | in vitro | iPSCs | Δ32 macrophages showed resistance to HIV [155]. |
| CRISPR-Cas9 knockout | Ablated the CCR5 gene and placed X4 infected cells under ganciclovir-augmented suicidal control | in vitro | TZM cells, H7 cells | CCR5 knock-out and Tat-activated conditional X4 inhibition conferred complete resistance to HIV [156]. |
| CRISPR-Cas9 knockout | Deletion of a 198-bp CCR5 fragment from exon 2 | in vitro | iPSCs from primate T cells and fibroblasts | Knockout cells prevented replication of the CCR5 tropic- SIVmac239 and -SIVmac316 [157]. |
| CRISPR-Cas9 knock-in | Induced a frameshift insertion in the CCR5 gene | in vitro | HSPCs, SCs, CD4+ T cells | Knock-ins disrupted CCR5 and conferred HIV resistance [158]. |
| CRISPR-Cas9 knockout | Induced CCR5 and CXCR4 single and dual knockouts | humice | Human CD4+T cells and PBMCs | Single knockouts resulted in HIV resistance, and dual knockouts inhibited CD4+T cells engraftment in BM [159]. |
| Base editing | evoCDA-BE4max converted CAA, CAG, CGA, and TGG to stop codons. ABE, using different PAM-specific nCas9s, showed broader coverage in mutating start codons | in vitro | HSPCs, Human CD4+ T cells | CBE and ABE complexes inhibited R5-tropic strains. CBE also inhibited X4 tropic strains [112]. |
| Prime editing | PE3max and uPEn converted C-T, disrupting CCR5 | in vitro | HEK293T cells | uPEn significantly reduced indel frequencies and ablated CCR5 [120]. |
TALENs, Transcription activator-like effector nucleases; HDR, Homology-directed repair; hu, humanized; NHP, non-human primate; NHEJ, Nonhomologous end joining; CBE, Cytosine base editor; ABE, Adenine base editor