Selectable one-step PCR-mediated integration of a degron for rapid depletion of endogenous human proteins.
Selectable one-step PCR-mediated integration of a degron for rapid depletion of endogenous human proteins.
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DOI：
10.2144/000114378
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发表时间：
2016-02
期刊：
影响因子：
2.7
通讯作者：
Bentley DL
中科院分区：
文献类型：
--
作者：
Sheridan RM;Bentley DL
通过配体调节的变性融合来控制蛋白质的稳定性是研究基因功能的一种强有力的方法。我们利用CRISPR/Cas9基因组编辑技术开发了一种可选择的盒，用于使用大肠杆菌二氢叶酸还原酶(EDHFR)降解子轻松地标记内源性人类蛋白的C末端。这种盒允许使用框内自裂解的2A肽和嘌呤霉素抗性基因高效地恢复正确的整合事件。PCR扩增的供体eDHFR盒片段两端各有100个碱基的同源性，通过引导RNA(GRNA)靶向双链DNA断裂的同源定向修复(HDR)整合在开放阅读框架(ORF)的3‘端。作为原理的证明，我们建立了三个内源性蛋白标记了eDHFR degron的细胞系。当抗生素甲氧苄啶从培养基中移除时，每个eDHFR标记的蛋白在2-4小时内都被消耗了90%，而这种消耗可以通过重新加入甲氧普林来逆转。由于嘌呤霉素选择允许仅使用100bp的同源长区域高效地恢复框内退化融合，该方法应该适用于全基因组范围的条件突变细胞系文库的构建。
Manipulation of protein stability with ligand-regulated degron fusions is a powerful method for investigating gene function. We developed a selectable cassette for easy C-terminal tagging of endogenous human proteins with the E. coli dihydrofolate reductase (eDHFR) degron using CRISPR/Cas9 genome editing. This cassette permits high-efficiency recovery of correct integration events using an in-frame self-cleaving 2A peptide and the puromycin resistance gene. PCR amplified donor eDHFR cassette fragments with 100 bases of homology on each end are integrated by homology-directed repair (HDR) of guide RNA (gRNA)-targeted double-stranded DNA breaks at the 3′ ends of open reading frames (ORFs). As proof of principle, we generated cell lines in which three endogenous proteins were tagged with the eDHFR degron. When the antibiotic trimethoprim is removed from the media, each of the eDHFR-tagged proteins was depleted by >90% within 2–4 h, and this depletion was reversed by re-addition of trimethoprim. Since puromycin selection permits recovery of in-frame degron fusions with high efficiency using only 100-bp long regions of homology, this method should be applicable on a genome-wide scale for generating libraries of conditional mutant cell lines.