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

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

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Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.
Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Kanagawa, Japan.
Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Developmental Neuroscience, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, Nebraska, USA.
The Institute of Medical Sciences, Tokai University, Kanagawa, Japan.


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.

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