title: Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.
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.
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, Center for Retrovirus Research, The Ohio State University Medical Center, Columbus, Ohio, USA.
authors.forename: Kristine E
authors.initials: KE
authors.lastname: Yoder
authors.affiliation: Department of Physics, Department of Chemistry and Biochemistry, Division of Hematology, Department of Internal Medicine, Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bundschuh
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053903:DNA Breaks, Double-Stranded; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting; D015658:HIV Infections / Q000235:genetics / Q000628:therapy*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D014779:Virus Replication
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems