Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.
Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.
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DOI：
10.1007/s10126-016-9708-6
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发表时间：
2016-08
影响因子：
3
通讯作者：
Collet, Bertrand
中科院分区：
文献类型：
--
作者：
Dehler, Carola E.;Boudinot, Pierre;Martin, Samuel A. M.;Collet, Bertrand
CRISPR/Cas9系统已广泛应用于动物和植物中指导诱变。迄今为止，还没有这样的方法存在于鱼体细胞系中。我们描述了在奇努克鲑鱼Oncorhynchus tshawytscha CHSE基因组编辑的有效程序。该细胞系经过基因修饰，首先过表达单个形式的EGFP（细胞系CHSE-E耐基因），另外过表达Cas9的核版本nCas9n（细胞系CHSE-EC， Hygromycin和Geneticin耐基因）。体外制备了预验证的sgRNA，并用于转染CHSE-EC细胞。通过流式细胞仪和显微镜检测，34.6%的细胞中EGFP基因被破坏。对目标位点进行PCR扩增、克隆及PCR产物测序鉴定；在25%的克隆中，EGFP基因通过预期位点的缺失而失活。该方法为高通量筛选相容的功能基因组研究开辟了前景。
CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.
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影响因子：
7
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
通讯作者：
Del Bene F
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
DOI：
10.1242/dev.115584
发表时间：
2014-12
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Irion U;Krauss J;Nüsslein-Volhard C
通讯作者：
Nüsslein-Volhard C
影响因子：
3.1
作者：
Lester, Katherine;Hall, Malcolm;Collet, Bertrand
通讯作者：
Collet, Bertrand
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle