Table 1 CRISPR/Cas Systems for targeting proviral HIV-1 (In-vitro studies)
CRISPR/Cas System | Cell type | Target region | Delivery | Results | References |
|---|---|---|---|---|---|
SpCas9 | HeLa, HEK293T, Jurkat | T5 site of TAR seq in the R region T6 site of NF-kB seq of the U3 region | Transfection | 45.6% to 20% decrease in proviral gene expression in 293Â T cells receiving T5 gRNA. Target site showed indel mutations | Ebina et al. [21] |
SpCas9 | HEK293T | LTR (R region) | Lentivirus | Decrease in protein expression regardless of the amount of integrated viral DNA | Liao et al. [47] |
SpCas9 | TZM.B1 | LTR | Transfection | Cleavage of viral DNA | Kaminski et al. [48] |
SpCas9 | CD4 + T cells PBMC (From patients) | LTR | Transfection | Decrease in viral cDNA number; Reduction in viral particles and expression of p24 and Gag proteins | Kaminski et al. [48] |
SpCas9 | CHME5, TZM-Bl, U937 | LTR | Transfection | gRNA-Cas9 complex excised a 9709 bp sequence between 5’ and 3’ LTR sequences | Hu et al. [49] |
SpCas9 | Jurkat (JLat 10.6) | LTR region, pol, rev (2nd exon) | Transfection | tenfold GFP reduction and 20-fold p24 reduction according to the respective gRNAs | Zhu et al. [50] |
SpCas9 | HeLa, Jurkat, TZM-bl | LTR (NF-κB Binding Sites) | Transfection Lentivirus | DSBs observed via GUIDE-seq, absence of off-target activity, reduction of 5’ LTR-driven HIV-1 transcription | Chung et al. [51] |
SpCas9 | Jurkat | LTR and viral genes | Lentivirus | Reduction of viral replication with dual gRNAs | Lebbink et al. [52] |
SpCas9 | HEK293T, SupT1 T cells | Gag, tat/rev, env | Lentivirus | Dual gRNAs treatment led to either of the three: hypermutation, excision or inversion | Binda et al. [53] |
SpCas9 | MT-4Â T cells HEK293T | Tat/Rev | Lentivirus | gRNA multiplexing against Tat in T cell line suppressed viral p24 protein and inhibited viral replication | Ophinni et al. [54] |
SpCas9 | 293Â T cells | LTR (R and U5) | Transfection | Three to five-fold reduction in integrated viral DNA, two-fold in late DNA and no change in early DNA | Yin et al. [55] |
SpCas9 | CD4 + T Cells Monocytes HEK 293FT Jurkat, ACH2 T cells | Exons (tat1-2, rev1-2, and gp41) | Transfection Electroporation Lentivirus Lipid nanoparticle (LNP) | Multi-exon gRNA TatDE delivered by LNPs showed 100% viral excision | Herskovitz et al. [44] |
SaCas9 | Jurkat C11 cells TZM-bI | LTR and viral genes | Lentivirus | Combination of SaCas9/gRNAs disrupted the HIV-1 genome more efficiently than a single sgRNA/SaCas9. Dual or Triple gRNAs in an all-in-one lentiviral vector reduced viral production | Wang Q et al. [56] |
Cas12a | HEK293T, SupT1 T cells | LTR | Lentivirus | Cas12a shows superior antiviral activity, achieve full HIV inactivation with only a single gRNA | Gao et al. [30] |
SpCas9 Cas12a (Transient) | SupT1 T cells | Gag, tat/rev | Lentivirus | Complete inactivation of proviral HIV-1 with repeated transfection of different Cas9 and Cas12a mRNA/protein sources with dual gRNAs | Liu et al. [46] |
Cas13d | CD4 + T cells | Gag, pol, protease, integrase | Lentivirus | Effectively inhibited HIV-1 infection and also, suppressed reactivated HIV-1 from latently infected cells | Nguyen et al. [31] |
Cas13a | HEK293T, JLat 10.6 | HIV-1 RNA | Lentivirus | Reduction in viral gene expression. Not only inhibits the newly synthesized viral RNA from the proviral DNA but also targets the viral RNA that enters host cells | Yin L et al. [58] |