Easi-CRISPR和TARGATT介导的鸡基因组安全位点功能验证

CRISPR/Cas9是一种强有力基因组编辑技术。但是，禽类还缺乏与哺乳动物Rosa26类似的安全位点, 限制了CRISPR/Cas9 knock-in技术在家禽转基因领域的应用。本研究首先用PhiC31整合酶系统在细胞水平上筛选整合效率最高的两个假attP位点, 再结合Easi-CRISPR和TARGAAT技术, 使Easi-CRISPR元件转染DF-1细胞或显微注射2.5日龄鸡胚背主动脉, 将PhiC31整合酶attPTT和attPCT位点锚定在这两个假attP位点上。通过基因分型（genotyping）和PhiC31介导的体外和体内基因盒交换, 证明这两个假attP位点的有效性；结合G1和G2转基因鸡繁殖能力和转基因遗传稳定性评估，证明其安全性，至少确定1个假attP位点为安全位点, 可望用于旨在制作动物疾病模型、抗病育种或提高生产性能为目的的条件性表达Cas9转基因鸡生产。

CRISPR/Cas9 is a powerful tool for gene editing. However, the application of CRISPR/Cas9 knock-in is limited in avian because of little knowledge about their genome safe harbors, some locus like Rosa26 in mammalian animals. The present study is to screen two pseudo attP sites with higher integration in chicken cells by means of PhiC31 integrase system, then have attPTT and attPCT landed in these two pseudo attP sites through combination of Easi-CRISPR and TARGAAT, via delivery of Easi-CRISPR reagents into DF-1 cells by in vitro transfection or in vivo micro injection to dorsal aorta of 2.5d chicken embryos. By means of genotyping and PhiC31 integrase mediated cassette exchange, the effectiveness of these two pseudo attP sites is to be confirmed, their safety is to be verified by determining the reproductive performance of G1 and G2 transgenic offsprings, and by detecting the genetic stability of the transgene. Finally, at least one of two pseudo attP sites is to be determined as a genome safe harbor, which could be available in production of transgenic chicken with conditional expression of Cas9, especially for animal disease models, anti-disease breeding and improvement of production traits.