Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9

Sci Rep. 2016 Jul 12;6:29530. doi: 10.1038/srep29530.

Abstract

CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CD4-Positive T-Lymphocytes / virology
  • CRISPR-Cas Systems*
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA Breaks, Double-Stranded
  • Gene Editing / methods*
  • Gene Targeting
  • HIV Infections / genetics
  • HIV Infections / therapy*
  • HIV-1 / genetics*
  • Humans
  • Mutation
  • RNA, Guide / genetics
  • Virus Replication

Substances

  • RNA, Guide