Format

Send to

Choose Destination
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.

Author information

1
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.
2
Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Kanagawa, Japan.
3
Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, Nebraska, USA.
4
Developmental Neuroscience, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, Nebraska, USA.
5
The Institute of Medical Sciences, Tokai University, Kanagawa, Japan.

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 ∼2 months to generate the founder mice.

PMID:
29266098
PMCID:
PMC6058056
DOI:
10.1038/nprot.2017.153
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center