Research Article: Gene Editing Approaches against Viral Infections and Strategy to Prevent Occurrence of Viral Escape

Date Published: December 8, 2016

Publisher: Public Library of Science

Author(s): Martyn K. White, Wenhui Hu, Kamel Khalili, Carolyn B Coyne.


Partial Text

Powerful new gene editing techniques promise groundbreaking opportunities for novel therapeutic options to important illnesses, including cancer, genetic disorders [1], and viral infections [2]. These techniques include zinc finger nucleases (ZFN) [3], transcription activator-like effector nucleases (TALEN) [4], and clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) [5, 6]. In particular, CRISPR/Cas9 provides an effective, highly specific, and versatile tool applicable to important human viruses, including HIV-1 [7]. CRISPR/Cas9 is elegant and simple compared to ZFN and TALEN because it uses one or more guide RNAs (gRNA), which are simple to produce and specifically target any sequence in an adaptable and flexible way for different targets, such as viral genes, by changing the gRNA sequence [8].

Genetic editing applications, including CRISPR/Cas9, can disrupt both episomal and integrated DNA viruses and can be applied to several human viruses [2], including papillomaviruses HPV16 and HPV18 [11–13], hepatitis B virus (HBV) [14–17], Epstein-Barr virus (EBV) [18, 19], HIV-1 [7, 20–27], polyomavirus JC (JCV) [28], Herpes simplex virus-1 [29, 30], and other herpesviruses [30].

Several reports have highlighted a caveat in CRISPR/Cas9 that is important to address when choosing gene editing strategy. CRISPR/Cas9 targeting of HIV-1 generated mutant viruses able to escape and replicate [39–42], as observed earlier with RNAi approaches [43–45]. One report described a mutant HIV-1 generated after ZFN therapy [39], and other reports indicated CRISPR/Cas9 gave profound suppression of HIV replication, but escape mutations were rapidly and consistently generated [40–42]. Escape mutants arose by insertions, deletions, and substitutions (InDels) located within the target site for Cas9 cleavage and are typical for DSBs repaired by NHEJ [40]. Cas9 cleavage inactivates the virus by introduction of mutations by NHEJ, but a subset of these retain viability and escape and are no longer susceptible to the original gRNA. Interestingly, while most InDels contributing to escape at non-coding regions were a single base pair, three base pair InDels were observed when the target was within an HIV-1 coding region; i.e., the InDel event may preserve the HIV-1 open reading frame but destroy the CRISPR gRNA sequence homology [42]. The occurrence of InDel escape mutations is a consequence of NHEJ DNA repair and so may also occur for any DNA virus or retrovirus and, indeed, was recently reported for the pseudorabies herpesvirus [46]. NHEJ is almost always the dominant mode of DNA repair [47, 48] and is the desired pathway of repair of CRISPR/Cas9-generated DSBs, because the purpose of CRISPR/Cas9 is to introduce mutations and inactivate viruses. It is also important to note that DSB and NHEJ repairs may have very different rates in some cells as compared to other cell types; e.g., T-cells relative to myeloid cells [49, 50]. However, as noted above, in studies of HIV-1 provirus excision, no cell-type-specific differences were observed.

Viral escape is not insurmountable if an appropriate choice of gene editing strategy is adopted. InDels introduced by NHEJ following CRISPR/Cas9 cleavage can cause frameshifts or premature stop codons, disrupting the target gene and abrogating its function and viral viability. If a unique locus is targeted, there is a significant possibility that InDels will be generated that allow viral escape; however, if multiplex gRNAs are employed, then the probability of this is greatly reduced, because the chances of two or more viability-conferring mutations are much less, as seen for mutiplex RNA interference (RNAi) [51]. Alternatively, if two gRNAs produce DNA breaks, allowing excision of a large section of DNA, this will permanently prevent the occurrence of escape mutations, as shown in Fig 1. Several studies have demonstrated the strong suppression of HIV-1 using a multiplex approach [21–25]. Table 1 summarizes recent studies [52–75] on the use of CRISPR/Cas9 technology for editing several human viruses using single and multiplex gRNAs, resulting in the introduction of InDels and/or excision of the segment of the viral genome.




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