HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA in each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection. The results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA during reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

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

“…Using experimental systems that support HIV replication, we and others demonstrated that HIV replication can be inhibited with Cas9 and a single antiviral gRNA. However, the virus can escape from this inhibition through the acquisition of specific mutations at the target site that prevent gRNA binding, but do not block virus replication [22][23][24][25][26]. Nucleotide substitutions were frequently observed when targeting highly conserved essential proviral sequences, whereas indels were selected when targeting less conserved regions that more easily tolerate larger mutations [22].…”

Endogenous Retroviruses Walk a Fine Line between Priming and Silencing

“…Due to the high mutation rate of retroviruses but conservation of the PBS, several studies suggested that destroying the PBS is the ultimate “cure” to disable LTR-retroelements [ 138 , 148 ]. This strategy is promising to target infectious retroviruses like HIV in somatic tissues but would come at a high cost in mammalian stem cells that have co-opted ERVs for cellular functions.…”

“…However, complementarity to the first 6 nt of the PBS has been sufficient for HIV-1 priming if compensated by additional interactions outside the PBS [ 147 ]. A recent study examined spontaneous mutations in HIV-1 after clustered regularly interspaced short palindromic repeat (CRISPR)-editing of the PBS [ 138 ]. Of note, HIV-1 repression is more persistent when the PBS is targeted on the minus strand because of the asymmetrical inheritance of the PBS in Retroviridae [ 138 ].…”

“…A recent study examined spontaneous mutations in HIV-1 after clustered regularly interspaced short palindromic repeat (CRISPR)-editing of the PBS [ 138 ]. Of note, HIV-1 repression is more persistent when the PBS is targeted on the minus strand because of the asymmetrical inheritance of the PBS in Retroviridae [ 138 ]. When targeting the plus strand PBS, the virus quickly evolves to avoid a perfect PBS.…”

“…Mismatches with the tRNA sequence are shaded in grey and black. Blue shaded nucleotide positions have been tolerant for mismatches when tested for the human immunodeficiency virus (HIV)-1, which uses tRNA Lysine 3 to prime reverse transcription [ 138 ]. ERVs that were active in retrotransposition assays are denoted with their names on the right, next to their respective PBS sequence [ 114 , 115 , 131 ].…”