CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA

Abstract

Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target sites correlated with permanent virus inactivation. To better understand the mechanism underlying HIV inactivation, we analyzed the proviral DNA upon Cas9 attack with gRNA pairs. We observed that dual-gRNA targeting resulted more frequently in target site mutation than fragment excision, while fragment inversion was rarely observed. The frequencies varied for different gRNA combinations without an obvious relationship with the distance between the target sites, indicating that other gRNA and target DNA characteristics influence the DNA cleavage and repair processes.

CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells

“…To explain this observation, we considered several possibilities that can lead to imperfect genomic Cas9 deletions in cells. Previous reports described that applications of the dual gRNA system (I) could lead to heterozygous deletion clones (8) whereby one allele carries the deletion and the other allele is unmodified, (II) mutations could have occurred at the PAM sequences (44, 45) preventing the recruitment of Cas9 and its cutting activity at the gene locus, (III) the Cas9 cleavage might be unsynchronized at the two DSB sites (46), in which one DSB would have been repaired before the induction of the second DSB or the target sequence could have been excised and then either (IV) inverted or (V) duplicated (8, 13, 47–49). Scenarios I to IV can be excluded since we would have detected a PCR product corresponding to the size of the unmodified allele (ca.…”

Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects

“…Thus, the dual-sgRNA combinations, consisting of two efficient and safe sgRNAs located between hundreds of bases in a protein-coding gene of HIV-1, could become a desirable and popular strategy to destroy the viral genome. Nonetheless, the low incidence of fragment excision and fragment inversion was an important issue that requires further attention ( Binda et al, 2020 ). In addition to the dual-sgRNAs CRISPR treatment strategy, according to a recent study ( Dash et al, 2019 ), a combination therapy comprising long-acting antiviral drugs and CRISPR treatment targeting HIV-1 DNA could lead to the safe elimination of HIV-1 infection, possibly representing a future cure for HIV-1.…”

The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

“…Although Cas9/multiplexed-sgRNA technology has emerged, the use of CRISPR/Cas9 molecular scissor to precisely and jointly target two coreceptor genes, CCR5 and CXCR4, has not yet been seen in relevant reports. Here, Table 1 lists the research studies of HIV-1 infection via CRISPR/Cas9 techniques for editing the aforementioned gene sites (Cho et al, 2013;Ebina et al, 2013;Hu et al, 2014;Hou et al, 2015; Wang et al, 2014;Ye et al, 2014;Liao et al, 2015;Zhu et al, 2015;Kaminski et al, 2016;Soppe and Lebbink, 2017;Liu et al, 2018;Kaushik et al, 2019;Binda et al, 2020). At present, purging of the latent viral reservoirs is the biggest hurdle for the effective management of HIV infection.…”

“…Many viruses possess the capability to escape or inhibit the effect of pharmaceuticals (e.g., interferon) in their evolution. Specifically in CRISPR/Cas9 applications, viruses can escape from these suppressions through the acquisitions of specific mutations at the target site that prevent gRNA binding without hindering viral replications (Binda et al, 2020). Using the HIV-1 as example, as far as we know, the RNA interference (RNAi) technology for the treatment of HIV-1 has already reached the clinical stage (Bobbin et al, 2015;Swamy et al, 2016).…”