High Levels of AAV Vector Integration Into CRISPR-induced DNA Breaks

Nat Commun. 2019 Sep 30;10(1):4439. doi: 10.1038/s41467-019-12449-2.

Abstract

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.

Publication types

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

MeSH terms

  • Animals
  • Bacteriophage lambda / genetics
  • Brain
  • CRISPR-Cas Systems*
  • Cell Line
  • Chromosome Mapping
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Cochlea
  • DNA Breaks*
  • Dependovirus / genetics*
  • Endonucleases
  • Gene Editing / methods*
  • Gene Targeting / methods
  • Genetic Therapy / methods
  • Genetic Vectors*
  • Genome
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Muscles
  • Neurons / virology
  • Targeted Gene Repair / methods
  • Treatment Outcome
  • Virus Integration / genetics*

Substances

  • Endonucleases