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

Hiromi Miura, Rolen M. Quadros, Channabasavaiah B Gurumurthy, Masato Ohtsuka

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

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 â 1/42 months to generate the founder mice.

Original languageEnglish (US)
Pages (from-to)195-215
Number of pages21
JournalNature protocols
Volume13
Issue number1
DOIs
StatePublished - Jan 1 2018

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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