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


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.

SUBMITTER: Miura H 

PROVIDER: S-EPMC6058056 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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

Miura Hiromi H   Quadros Rolen M RM   Gurumurthy Channabasavaiah B CB   Ohtsuka Masato M  

Nature protocols 20171221 1


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  ...[more]

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