CRISPR and associated Cas nucleases are genetic engineering tools revolutionizing innovative approaches to cancer and inherited diseases. CRISPR-directed gene editing relies heavily on proper DNA sequence alignment between the guide RNA (gRNA)/CRISPR complex and its genomic target. Accurate hybridization of complementary DNA initiates gene editing in human cells, but inherent gRNA sequence variation that could influence the gene editing reaction has been clearly established among diverse genetic populations. As this technology advances toward clinical implementation, it will be essential to assess what degree of gRNA variation generates unwanted and erroneous CRISPR activity. With the use of a system in which a cell-free extract catalyzes nonhomologous end joining (NHEJ) and homology-directed repair (HDR), it is possible to observe a more representative population of all forms of gene editing outcomes. In this manuscript, we demonstrate CRISPR/Cas complexation at heterologous binding sites that facilitate precise and error-prone HDR. The tolerance of mispairing between the gRNA and target site of the DNA to enable HDR is surprisingly high and greatly influenced by polarity of the donor DNA strand in the reaction. These results suggest that some collateral genomic activity could occur at unintended sites in CRISPR-directed gene editing in human cells.

CRISPR 和相关的 Cas 核酸酶是基因工程工具，彻底改变了癌症和遗传性疾病的创新方法。 CRISPR 指导的基因编辑在很大程度上依赖于引导 RNA (gRNA)/CRISPR 复合物与其基因组靶标之间正确的 DNA 序列比对。互补 DNA 的精确杂交启动了人类细胞中的基因编辑，但在不同的遗传群体中已经明确确定了可能影响基因编辑反应的固有 gRNA 序列变异。随着这项技术向临床实施迈进，评估 gRNA 变异的程度会产生不需要的和错误的 CRISPR 活性至关重要。通过使用无细胞提取物催化非同源末端连接（NHEJ）和同源定向修复（HDR）的系统，可以观察到所有形式的基因编辑结果中更具代表性的群体。在这份手稿中，我们展示了异源结合位点上的 CRISPR/Cas 络合，这有利于精确且容易出错的 HDR。 gRNA 和 DNA 靶位点之间错配的耐受性（使 HDR 成为可能）出人意料地高，并且很大程度上受反应中供体 DNA 链极性的影响。这些结果表明，在人类细胞中 CRISPR 指导的基因编辑中，一些附带的基因组活性可能发生在非预期位点。