title: Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9.
abstract: 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.
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China; Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, Changchun, China.
authors: Luqing
authors: L
authors: Zhang
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Ruirui
authors: R
authors: Jia
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Norberto J
authors: NJ
authors: Palange
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Achim Cchitvsanzwhoh
authors: AC
authors: Satheka
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Jacques
authors: J
authors: Togo
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Yao
authors: Y
authors: An
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Mabwi
authors: M
authors: Humphrey
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Luying
authors: L
authors: Ban
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Yan
authors: Y
authors: Ji
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.
authors: Honghong
authors: H
authors: Jin
authors: Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, Changchun, China.
authors: Xuechao
authors: X
authors: Feng
authors: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China; Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, Changchun, China.
authors: Yaowu
authors: Y
authors: Zheng
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter / Q000235:genetics; D016678:Genome / Q000235:genetics*; D059765:Homologous Recombination / Q000235:genetics; D008297:Male; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D015053:Zygote / Q000378:metabolism