CRISPR/Cas9 and Genome Editing for Viral Disease-Is Resistance Futile?

Abstract

Chronic viral infections remain a major public health issue affecting millions of people worldwide. Highly active antiviral treatments have significantly improved prognosis and infection-related morbidity and mortality but have failed to eliminate persistent viral forms. Therefore, new strategies to either eradicate or control these viral reservoirs are paramount to allow patients to stop antiretroviral therapy and realize a cure. Viral genome disruption based on gene editing by programmable endonucleases is one promising curative gene therapy approach. Recent findings on RNA-guided human immunodeficiency virus 1 (HIV-1) genome cleavage by Cas9 and other gene-editing enzymes in latently infected cells have shown high levels of site-specific genome disruption and potent inhibition of virus replication. However, HIV-1 can readily develop resistance to genome editing at a single antiviral target site. Current data suggest that cellular repair associated with DNA double-strand breaks can accelerate the emergence of resistance. On the other hand, a combination antiviral target strategy can exploit the same repair mechanism to functionally cure HIV-1 infection in vitro while avoiding the development of resistance. This perspective summarizes recent findings on the biology of resistance to genome editing and discusses the significance of viral genetic diversity on the application of gene editing strategies toward cure.

Keywords: CRISPR resistance; CRISPR/Cas9; HIV; genetic diversity; hepatitis B virus; herpes simplex virus.

Figure 1. Pathways to HIV endonuclease resistance

Endonuclease-mediated cleavage of the HIV provirus leads to NHEJ-dependent mutation that produces either replication-deficient or endonuclease-resistant and replication competent mutant progeny. Endonuclease-resistant genomes may also be present within global HIV variants, within the host viral quasispecies, or can be generated through direct hypermutations of viral RNA by APOBEC proteins, reverse transcription mutations during provirus synthesis, or RNA pol II mutations introduced during viral RNA synthesis.

Figure 2. HIV sequence diversity

a, Phylogenetic trees representing HIV diversity were made using the Los Alamos National Laboratory (LANL) 2016 HIV complete genome alignments for all HIV sequences (left) or M group A–K genomes without recombinants (right). Trees were generated with Geneious using the Neighbor-Joining method and a Jukes-Cantor genetic distance model. Scale represents nucleotide substitutions per site. b, nucleotide diversity plots for HIV complete genomes, LTR, gag, pol and env, generated using data from the LANL 2016 M group alignment. Nucleotide conservation is represented in terms of information content (range = 0–2 bits) calculated with a moving window width of 50 nt.