Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), German Cancer Research Center (DKFZ)-ZMBH Alliance, Heidelberg, Germany.
Center for Organismal Studies, University of Heidelberg and DKFZ, DKFZ-ZMBH Alliance, Heidelberg, Germany.
J Cell Biol. 2020 Jun 1;219(6). doi: 10.1083/jcb.201910210.
Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.
在这里，我们描述了一种在哺乳动物组织培养细胞中进行内源性 C 端基因标记的高效策略。我们使用在线平台设计两个用于 PCR 的长基因特异性寡核苷酸，与通用模板盒一起创建线性 dsDNA 供体，称为 PCR 盒。PCR 盒编码标签（例如 GFP）、Cas12a CRISPR RNA，用于切割靶基因座，以及短同源臂，用于通过同源重组进行定向整合。整合的标签与通用终止子相连，该终止子屏蔽了标记基因与插入的辅助序列。将 PCR 盒与 Cas12a 编码质粒共转染可导致标记基因的内源性表达增强，在没有选择的情况下标记效率高达 20%，使用选择标记时高达 60%。我们使用目标富集测序来研究所有潜在的人工制品来源。我们的工作概述了一种快速策略，特别适合使用荧光蛋白标记基因的内源性表达进行探索性研究。
