Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors

Nat Protoc. 2018 Jan;13(1):195-215. doi: 10.1038/nprot.2017.153. Epub 2017 Dec 21.


CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • DNA, Single-Stranded / genetics*
  • Female
  • Gene Editing / methods*
  • Gene Knock-In Techniques / methods*
  • Gene Knockout Techniques / methods*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout


  • DNA, Single-Stranded