Chakrapani Vemulawada, Patra Swagat Kumar, Panda Rudra Prasanna, Rasal Kiran Dashrath, Jayasankar Pallipuram, Barman Hirak Kumar
Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India.
Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India.
Dev Comp Immunol. 2016 Aug;61:242-7. doi: 10.1016/j.dci.2016.04.009. Epub 2016 Apr 11.
Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes.
基因编辑技术的最新进展尚未在养殖鱼类中得到应用。我们利用CRISPR/Cas9系统在养殖鲤鱼（印度魮，俗称露斯塔野鲮）中建立了一种基因靶向技术。我们证明，供体DNA通过同源重组（HR）整合到由CRISPR/Cas9核酸酶产生的靶向双链切口位点。这导致成功破坏了印度魮Toll样受体22（TLR22）基因，该基因参与先天免疫，仅存在于硬骨鱼类和两栖动物中。由此产生的无效突变体缺乏TLR22 mRNA表达。总之，这是首个证据表明CRISPR/Cas9系统是通过硬骨鱼类中的同源重组进行靶向基因破坏以生成大型养殖鱼类模型的高效工具。
