Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA.
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA.
Nat Commun. 2017 Nov 23;8(1):1711. doi: 10.1038/s41467-017-01875-9.
Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene-editing methods, and enrich cell populations containing multiplexed precise edits up to 42-fold. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro or ex vivo gene-editing applications in precision medicine and drug discovery and aid in the development of increased and serial dosing regimens for somatic gene editing in vivo.
将特定的 DNA 序列写入人类基因组具有挑战性，因为大多数编辑的序列包含各种不精确的插入或缺失。我们开发了一种模块化的 RNA 适体-链霉亲和素策略，称为 S1mplex，用于将 CRISPR-Cas9 核糖核蛋白与核酸供体模板以及其他生物素化分子（如量子点）复合。在人类细胞中，定制的 S1mplex 将精确编辑与不精确编辑等位基因的比例提高了 18 倍，高于标准的基因编辑方法，并将包含多重精确编辑的细胞群体富集提高了 42 倍。这些使用多功能、预组装试剂的进展可以大大减少精确医学和药物发现中体外或离体基因编辑应用的时间和成本，并有助于开发体内体细胞基因编辑的增加和连续剂量方案。
