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.

中文翻译：

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哺乳动物基因的 CRISPR-Cas12a 辅助 PCR 标记

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 C 末端基因标记的省时策略。在线平台用于设计两种用于 PCR 的长基因特异性寡核苷酸，并使用通用模板盒创建线性 dsDNA 供体，称为 PCR 盒。PCR 盒编码标签（例如 GFP）、用于切割靶基因座的 Cas12a CRISPR RNA 以及用于通过同源重组进行定向整合的短同源臂。整合的标签与通用终止子偶联，保护带标签的基因免受共同插入的辅助序列的影响。PCR 盒与 Cas12a 编码质粒的共转染可实现标记基因的稳健内源表达，在不进行选择的情况下，标记效率高达 20%，而在使用选择标记时，标记效率高达 60%。我们使用目标富集测序来调查所有潜在的伪影来源。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表达进行探索性研究。