Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.
Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.
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DOI：
10.1371/journal.pone.0154303
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Leighton PA
中科院分区：
文献类型：
--
作者：
Dimitrov L;Pedersen D;Ching KH;Yi H;Collarini EJ;Izquierdo S;van de Lavoir MC;Leighton PA
CRISPR/Cas9系统已经应用于大量动植物的基因组编辑。在鸡中，CRISPR已被用于敲除体细胞组织中的基因，但尚未有CRISPR介导的生殖系修饰的报道。在这里，我们使用CRISPR来靶向原始生殖细胞(PGCs)中的鸡免疫球蛋白重链基因，以产生转基因后代。将引导RNA与供体载体共转染，以进行双链断裂的同源定向修复，并选择克隆群体。所有产生的抗药性克隆都包含正确的靶向事件。靶向细胞产生了包含CRISPR靶向基因的健康后代。结果表明，用CRISPR/Cas9系统在体外修饰PGCs可以获得基因编辑鸡，为高效的禽类遗传修饰开辟了许多潜在的应用前景。
The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.