Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9
| Autor: | Jacques Togo, Honghong Jin, Ruirui Jia, Yao An, Xuechao Feng, Luqing Zhang, Yan Ji, Achim Cchitvsanzwhoh Satheka, Yaowu Zheng, Norberto J. Palange, Luying Ban, Mabwi Humphrey |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Male
Zygote Mutagenesis (molecular biology technique) lcsh:Medicine Biology Mice Plasmid Genes Reporter CRISPR Animals Gene Knock-In Techniques lcsh:Science Homologous Recombination Embryonic Stem Cells Genetics Transcription activator-like effector nuclease Reporter gene Multidisciplinary Genome Base Sequence Cas9 Point mutation lcsh:R Gene targeting Molecular biology Mutagenesis Insertional lcsh:Q Female CRISPR-Cas Systems Gene Deletion Research Article Plasmids |
| Zdroj: | PLoS ONE PLoS ONE, Vol 10, Iss 3, p e0120396 (2015) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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