Ghosh Sanjay, Liu Ji-Long
Department of Physiology, Anatomy, and Genetics, MRC Functional Genomics Unit, University of Oxford, Oxford, OX1 3PT, UK.
Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK.
Methods Mol Biol. 2018;1680:217-235. doi: 10.1007/978-1-4939-7339-2_15.
Tagging of genes at the endogenous loci is a powerful strategy for the analysis of protein function. We have developed a homologous recombination-based approach for inserting epitope tag into Drosophila AGO1 locus by employing the CRISPR/Cas9 technology. The methodology involves co-expression of sgRNA (containing 20-nucleotide AGO1 targeting sequence) and Cas9 protein, together with a donor template that has HA-AGO1 cassette flanked by sequences homologous to the AGO1 locus. The integration is efficient and readily monitored by immunostaining of the transgenic cell line. This method facilitates rapid generation of stable cell lines and allows insertion of any tag sequence into endogenous loci, thus accelerating characterization of the tagged proteins.
在内源基因座对基因进行标记是分析蛋白质功能的一种强大策略。我们开发了一种基于同源重组的方法，通过利用CRISPR/Cas9技术将表位标签插入果蝇AGO1基因座。该方法包括共表达sgRNA（包含20个核苷酸的AGO1靶向序列）和Cas9蛋白，以及一个供体模板，该模板具有HA-AGO1盒，其两侧是与AGO1基因座同源的序列。通过对转基因细胞系进行免疫染色，可以高效且容易地监测整合情况。这种方法有助于快速生成稳定的细胞系，并允许将任何标签序列插入内源基因座，从而加速对标记蛋白的表征。
