Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. Howev...

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Bibliographic Details
Published in:PloS one 2015-03, Vol.10 (3), p.e0120396-e0120396
Main Authors: Zhang, Luqing, Jia, Ruirui, Palange, Norberto J, Satheka, Achim Cchitvsanzwhoh, Togo, Jacques, An, Yao, Humphrey, Mabwi, Ban, Luying, Ji, Yan, Jin, Honghong, Feng, Xuechao, Zheng, Yaowu
Format: Article
Language:English
Subjects:
Animal models
Animals
Base Sequence
Clonal deletion
Coinjection
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
Dilution
DNA
Embryo cells
Embryonic stem cells
Embryonic Stem Cells - metabolism
Embryos
Female
Fragmentation
Galactosidase
Gene Deletion
Gene Knock-In Techniques
Genes, Reporter - genetics
Genome - genetics
Homologous Recombination - genetics
Injection
Insertion
Kinases
Male
Mice
Mutagenesis, Insertional - methods
Mutation
Plasmids
Plasmids - genetics
Reporter gene
Rodents
Stem cell transplantation
Stem cells
Transcription
Transfection
Zygote - metabolism
Zygotes
β-Galactosidase
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Summary:ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0120396