To realize the full potential of the CRISPR/Cas system and expand its applicability up to the detection of molecular interactions, we herein describe a novel method to identify protein/small molecule interactions by utilizing the CRISPR/Cas12a collateral cleavage activity. This technique employs a single-stranded activator DNA modified with a specific small molecule, which would switch on the CRISPR/Cas12a collateral cleavage activity upon binding to crRNA within the CRISPR/Cas12a system. When the target protein binds to the small molecule on the activator DNA, the bound protein sterically hinders the access of the activator DNA to crRNA, thereby promoting less collateral cleavage activity of CRISPR/Cas12a. As a consequence, fewer reporter probes nearby are cleaved to produce accordingly reduced fluorescence signals in response to target protein. Based on this unique design principle, the two model protein/small molecule interactions, streptavidin/biotin and anti-digoxigenin/digoxigenin, were successfully determined down to 0.03 nM and 0.09 nM, respectively, with a fast and simple detection workflow (11 min). The practical applicability of this method was also verified by reliably detecting target streptavidin spiked in heterogeneous human serum. This work would provide great insight to construct novel strategies to identify protein/small molecule interaction by making the most of the CRISPR/Cas12a system beyond its superior capabilities in genome editing and molecular diagnostics.

为了充分发挥 CRISPR/Cas 系统的潜力并将其适用性扩展到分子相互作用的检测，我们在此描述了一种利用 CRISPR/Cas12a 附带裂解活性识别蛋白质/小分子相互作用的新方法。该技术采用经特定小分子修饰的单链激活剂 DNA，在与 CRISPR/Cas12a 系统内的 crRNA 结合后，将开启 CRISPR/Cas12a 附带裂解活性。当靶蛋白与激活剂 DNA 上的小分子结合时，结合的蛋白质会在空间上阻碍激活剂 DNA 进入 crRNA，从而促进 CRISPR/Cas12a 较低的附带裂解活性。因此，附近较少的报告探针被切割以产生相应减少的响应靶蛋白的荧光信号。基于这种独特的设计原理，两种模型蛋白质/小分子相互作用，链霉亲和素/生物素和抗地高辛/地高辛，分别成功测定到 0.03 nM 和 0.09 nM，检测工作流程快速简单（11 分钟） . 该方法的实际适用性也通过可靠地检测掺入异质人血清中的目标链霉亲和素得到验证。通过充分利用 CRISPR/Cas12a 系统在基因组编辑和分子诊断方面的卓越能力，这项工作将为构建识别蛋白质/小分子相互作用的新策略提供深刻见解。分别具有快速简单的检测工作流程（11 分钟）。该方法的实际适用性也通过可靠地检测掺入异质人血清中的目标链霉亲和素得到验证。通过充分利用 CRISPR/Cas12a 系统在基因组编辑和分子诊断方面的卓越能力，这项工作将为构建识别蛋白质/小分子相互作用的新策略提供深刻见解。分别具有快速简单的检测工作流程（11 分钟）。该方法的实际适用性也通过可靠地检测掺入异质人血清中的目标链霉亲和素得到验证。通过充分利用 CRISPR/Cas12a 系统在基因组编辑和分子诊断方面的卓越能力，这项工作将为构建识别蛋白质/小分子相互作用的新策略提供深刻见解。