Genome-wide detection of CRISPR editing in vivo using GUIDE-tag

Nat Commun. 2022 Jan 21;13(1):437. doi: 10.1038/s41467-022-28135-9.


Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Biotin / metabolism
  • Biotinylation
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics*
  • Cell Line, Tumor
  • DNA / metabolism
  • Gene Editing*
  • Genes, Reporter
  • Genome
  • Liver / metabolism
  • Lung / metabolism
  • Mice
  • RNA, Guide, Kinetoplastida / genetics*
  • Ribonucleoproteins / metabolism


  • RNA, Guide
  • Ribonucleoproteins
  • Biotin
  • DNA
  • CRISPR-Associated Protein 9