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

• Published in: Scientific reports Vol. 6; no. 1; p. 29530
• Main Authors: Yoder, Kristine E., Bundschuh, Ralf
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.07.2016; Nature Publishing Group
• Subjects: 13; 13/106; 45/22; 45/23; 631/208; 631/326/596; CD4-Positive T-Lymphocytes - virology; Cell cycle; Cell Line; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Cas Systems; DNA Breaks, Double-Stranded; Gene Editing - methods; Gene Targeting; Genes; Genomes; HIV Infections - genetics; HIV Infections - therapy; HIV-1 - genetics; Humanities and Social Sciences; Humans; multidisciplinary; Mutation; Proteins; RNA polymerase; RNA, Guide, CRISPR-Cas Systems - genetics; Science; Virus Replication; Yeast; United States > US; Ohio
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep29530

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.