Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9

Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vecto...

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Bibliographic Details
Published in:Scientific reports 2015-06, Vol.5 (1), p.11221, Article 11221
Main Authors: Hara, Satoshi, Tamano, Moe, Yamashita, Satoshi, Kato, Tomoko, Saito, Takeshi, Sakuma, Tetsushi, Yamamoto, Takashi, Inui, Masafumi, Takada, Shuji
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Cell lines
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR
Deoxyribonucleases, Type II Site-Specific - metabolism
Deoxyribonucleic acid
DNA
Fusion protein
Genome editing
Genomes
gRNA
Mice
Mice, Mutant Strains
Microinjection
Molecular Sequence Data
mRNA
Mutagenesis
Nuclease
Rodents
Transcription activator-like effector nucleases
Zygotes
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Summary:Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vector construction and high efficiency of genome editing, it raises concerns of off-target effects when single guide RNA (gRNA) is used. Recently, FokI-dCas9 (fCas9), a fusion protein comprised of the inactivated mutant form of Cas9 and the DNA nuclease domain of FokI, has been developed. It enables genome editing with reduced risks of off-target effects in mammalian cultured cell lines, as fCas9 requires gRNAs to bind opposite strands with an appropriate distance between them. Here, we demonstrated that fCas9 efficiently generates living mutant mice through microinjection of its mRNA and gRNAs into zygotes. A comparison of the relative efficiencies of genome editing using fCas9 and other modified Cas9s showed that these mutagenesis efficiencies are similar when the targets of two gRNAs are separated by an appropriate distance, suggesting that in addition to the ease of vector construction, fCas9 exhibit high efficiency in producing mutant mice and in reducing risks of off-target effects.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep11221