Centre for Craniofacial and Regenerative Biology, King's College London, London SE1 9RT, U.K.
Department of Chemistry, Kongju National University, Gongju 32588, Republic of Korea.
ACS Appl Mater Interfaces. 2023 Nov 1;15(43):49964-49973. doi: 10.1021/acsami.3c07918. Epub 2023 Sep 28.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.
成簇规律间隔短回文重复序列（CRISPR）相关蛋白（Cas）（CRISPR/Cas）系统由于其简单性、敏感性、特异性和广泛适用性，最近已成为强大的分子生物传感工具，基于其旁切活性。然而，直接应用旁切活性进行原位细胞内检测仍然具有挑战性。在这里，我们首次推出了一种用于细胞内三磷酸腺苷（ATP）的 CRISPR/Cas 辅助纳米针传感器（nanoCRISPR），该传感器通过引入两步细胞内靶标识别过程，然后进行细胞外转导和检测，避免了与细胞内旁切相关的挑战。ATP 识别首先通过将与纳米针结合的与适体锁定的 Cas12a 激活剂呈现在细胞胞质溶胶中来发生；识别事件会解锁固定在纳米针上的激活剂。然后将纳米针从细胞中取出并暴露于 Cas12a/crRNA 复合物中，其中激活剂触发 ssDNA 荧光团猝灭剂对的切割，产生可检测的荧光信号。nanoCRISPR 的 ATP 检测限为 246 nM，动态范围为 1.56 至 50 μM。重要的是，nanoCRISPR 可以在 30 分钟内在活细胞中检测到细胞内 ATP，而不会影响细胞活力。我们预计，nanoCRISPR 方法将有助于拓宽 CRISPR/Cas 传感器在生物医学中的应用，用于检测活系统中各种细胞内分子。
