Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.
Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
Cell. 2019 May 2;177(4):1067-1079.e19. doi: 10.1016/j.cell.2019.04.009.
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
精确控制 CRISPR-Cas9 活性是许多基因组工程技术所必需的。在这里，我们报告了一个可推广的平台，该平台提供了第一个针对化脓性链球菌 Cas9（SpCas9）的合成小分子抑制剂，其分子量 <500Da，可穿透细胞，在生理条件下可还原且稳定。我们开发了一系列用于 SpCas9 功能的高通量测定法，包括用于 SpCas9 与前导序列相邻基序结合的初步筛选测定法，并使用这些测定法筛选了结构多样的天然产物样小分子文库，最终确定了可破坏 SpCas9-DNA 相互作用的化合物。使用这些合成抗 CRISPR 小分子，我们展示了 SpCas9 和具有催化缺陷的 SpCas9 技术的剂量和时间控制，包括转录激活，并使用结构活性关系确定了 SpCas9 抑制的药效团。这些研究建立了一个平台，用于快速鉴定针对 SpCas9 和下一代 CRISPR 相关核酸酶的合成、微型、可穿透细胞和可还原的抑制剂。
