Abstract：
Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.
摘要：
病毒介导的CRISPR-Cas9系统的递送是修饰细胞基因组最常用的技术之一。目的是分析目标基因产物的功能。虽然这些方法对于膜结合蛋白相当简单，它们对于细胞内蛋白质可能很费力，鉴于完全敲除（KO）细胞的选择通常需要扩增单细胞克隆。此外，病毒介导的递送系统，除了Cas9和gRNA,导致不需要的遗传物质的整合，比如抗生素抗性基因，引入实验偏见。这里,针对CRISPR/Cas9提出了一种替代的非病毒递送方法,允许有效和灵活地选择KO多克隆细胞.这种一体化的哺乳动物CRISPR-Cas9表达载体，ptARgenOM,编码与核糖体跳跃肽序列连接的gRNA和Cas9，然后是增强的绿色荧光蛋白和嘌呤霉素N-乙酰转移酶，允许瞬态，等基因KO细胞的表达依赖性选择和富集。在6个细胞系中使用超过12个不同的靶标进行评估后，发现ptARgenOM在生产KO细胞中是有效的，将获得多克隆等基因细胞系所需的时间减少4-6倍。AltogetherptARgenOM提供了一个简单的,快,以及用于基因组编辑的具有成本效益的交付工具。
