Baculovirus-based genome editing in primary cells

被引:18
作者
Mansouri, Maysam [1 ,2 ]
Ehsaei, Zahra [3 ]
Taylor, Verdon [3 ]
Berger, Philipp [1 ]
机构
[1] Paul Scherrer Inst, Biomol Res, Mol Cell Biol, CH-5232 Villigen, Switzerland
[2] ETH, Dept Biol, CH-8093 Zurich, Switzerland
[3] Univ Basel, Dept Biomed, CH-4058 Basel, Switzerland
基金
瑞士国家科学基金会;
关键词
Genome editing; CRISPR/Cas9; Baculovirus; Primary cells; CRISPR-CAS9; CRISPR/CAS9; EFFICIENCY; DELIVERY; ZYGOTES; KNOCKIN; SYSTEM;
D O I
10.1016/j.plasmid.2017.01.003
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS). (C) 2017 Elsevier Inc. All rights reserved.
引用
收藏
页码:5 / 9
页数:5
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