Hewes Amanda M, Sansbury Brett M, Barth Shaul, Tarcic Gabi, Kmiec Eric B
Gene Editing Institute, Helen F. Graham Cancer Center & Research Institute, Christiana Care Health System, Newark, DE, USA.
Gene Editing Institute, Helen F. Graham Cancer Center & Research Institute, Christiana Care Health System, Newark, DE, USA; Department of Medical and Molecular Sciences, University of Delaware, Newark, DE, USA.
Mol Ther Nucleic Acids. 2020 Jun 5;20:568-579. doi: 10.1016/j.omtn.2020.03.012. Epub 2020 Mar 30.
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及相关的Cas核酸酶是基因工程工具，正在彻底改变癌症和遗传性疾病的创新治疗方法。CRISPR导向的基因编辑严重依赖于向导RNA（gRNA）/CRISPR复合物与其基因组靶点之间正确的DNA序列比对。互补DNA的准确杂交启动人类细胞中的基因编辑，但在不同遗传群体中，已经明确存在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应用推进，评估何种程度的gRNA变异会产生不必要和错误的CRISPR活性至关重要。通过使用一种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，可以观察到更具代表性的所有形式基因编辑结果的群体。在本论文中，我们展示了在促进精确和易出错的HDR的异源结合位点处的CRISPR/Cas复合作用。gRNA与DNA靶位点之间错配以实现HDR的耐受性出奇地高，并且在反应中受到供体DNA链极性的极大影响。这些结果表明，在人类细胞中CRISPR导向的基因编辑过程中，一些附带的基因组活性可能会在非预期位点发生。
