CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

Abstract

Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications.

Figure 1

Cas9/gRNA targeting of the HIV-1 genome. (a) The HIV-1 proviral DNA with the position of gRNAs tested in this study. (b) The efficiency of gRNAs to silence HIV-1 DNA was tested in 293T cells transfected with plasmids expressing Cas9, gRNA, and HIV-1 LAI. To quantify viral gene expression, the viral capsid protein (CA-p24) was measured in the culture supernatant at 2 days after transfection. Average values (±SD) of four experiments are shown. Statistical analysis (independent samples' t-test analysis) demonstrated that CA-p24 expression in the presence of antiviral gRNAs differed significantly from values measured with control gRNAs against luciferase and GFP (*P < 0.05).

Figure 2

HIV-1 replication in Cas9 and gRNA expressing cells. (a,b) SupT1 cells stably transduced with Cas9 and gRNA expressing lentiviral vectors were infected with HIV-1 LAI. Virus replication was monitored by measuring the CA-p24 level in the culture supernatant (a) and by scoring the formation of virus-induced syncytia (b). The day at which massive syncytia were observed, which reflects breakthrough virus replication, is indicated. Average values of four experiments (±SD) are shown. SupT1, control nontransduced cells. SupT1-Cas9, cells transduced only with the Cas9 expressing vector. (c) Correlation between the level of inhibition (day of breakthrough replication; as shown in b and the conservation of target sequence amongst different HIV-1 isolates (Shannon entropy as shown in Supplementary Table S1). The Pearson's correlation coefficient was calculated: r = −0.58.

Figure 3

HIV-1 escapes from Cas9/gRNA inhibition through mutations in the target region. The gRNA target region in breakthrough viruses obtained in two to six independent HIV-1 cultures on the different SupT1-Cas9-gRNA cells was sequenced. For every gRNA, the wild-type HIV-1 nucleotide sequence is shown on top. Codons are boxed in grey if applicable, with the translated amino acid sequence on the right hand side. The protospacer adjacent motif (PAM) sequence is boxed and the arrowhead indicates the Cas9 cleavage site at position −3. Nucleotide and amino acid substitutions, insertions and deletions (Δ) are indicated. Data for all tested gRNAs are shown in Supplementary Figure S3.

Figure 4

HIV-1 escape mutations cluster around the Cas9 cleavage site. The position of all observed nucleotide deletions, insertions, and substitutions (as shown in Figure 3 and Supplementary Figure S3) was plotted relative to the gRNA target sequence (position −1 to −20) and PAM (position 1–3). The arrowhead indicates the Cas9 cleavage site at position −3.