CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called “sgR-DNA” that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA. Our sgR-DNA-based method is simple to implement because it does not require cloning steps. We also report the potential of our delivery system to generate multiplex genomic substitutions in Escherichia coli and human cells.

中文翻译：

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基于CRISPR / Cas9系统的编码sgRNA和供体DNA的线性化双链DNA的直接传递，用于生成单核苷酸变体

用于基因组编辑的CRISPR / Cas9需要提供指导RNA序列和供体DNA才能进行靶向的同源重组。通常，作为供体模板的单链寡聚脱氧核苷酸和编码指导RNA的质粒作为两个单独的组分递送。然而，在单核苷酸变体的多重产生中，这种两组分递送系统受到将匹配的sgRNA和供体DNA对递送至靶细胞的困难的限制。在这里，我们描述了一种新型的代码传递系统，称为“ sgR-DNA”，它使用由供体DNA成分和编码sgRNA的成分组成的线性化双链DNA。我们的基于sgR-DNA的方法易于实现，因为它不需要克隆步骤。我们还报告了我们的递送系统在产生多重基因组替代中的潜力。大肠杆菌和人类细胞。