Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, Shaanxi, P. R. China.
Anal Chem. 2022 Mar 8;94(9):3987-3996. doi: 10.1021/acs.analchem.1c05124. Epub 2022 Feb 22.
The precise and controllable programming of the -cleavage activity of the CRISPR-Cas13a systems is significant but challenging for fabricating high-performance biosensing systems toward various kinds of biomolecule targets. In this work, we have demonstrated that under a critical low Mg concentration, a simple and short single-stranded DNA (ssDNA) probe free of any modification can efficiently prevent the assembly of crRNA and LwaCas13a only by partially binding with the crRNA repeat region, thereby blocking the -cleavage activity of the LwaCas13a system. Furthermore, we have demonstrated that the blocked -cleavage activity of the LwaCas13a system can be recovered by various kinds of biologically important substances as long as they could specifically release the blocker DNA from the crRNA in a target-responsive manner, providing a facile route for the quantification of diverse biomarkers such as enzymes, antigens/proteins, and exosomes. To the best of our knowledge, this is reported for the first time that a simple ssDNA can be employed as the switch element to control the crRNA structure and regulate the -cleavage activity of Cas13a, which has enriched the CRISPR-Cas13a sensing toolbox and will greatly expand its application scope.
精确和可控的 CRISPR-Cas13a 系统的 -切割活性编程对于制造针对各种生物分子靶标的高性能生物传感系统具有重要意义，但也具有挑战性。在这项工作中，我们证明了在临界低镁浓度下，一种简单的短单链 DNA（ssDNA）探针，无需任何修饰，仅通过部分结合 crRNA 重复区域，就可以有效地阻止 crRNA 和 LwaCas13a 的组装，从而阻断 LwaCas13a 系统的 -切割活性。此外，我们证明，只要各种生物重要物质能够以靶标响应的方式特异性地从 crRNA 上释放出阻断 DNA，被阻断的 LwaCas13a 系统的 -切割活性就可以被恢复，为各种生物标志物（如酶、抗原/蛋白和外泌体）的定量提供了一种简单的方法。据我们所知，这是首次报道一种简单的 ssDNA 可被用作开关元件来控制 crRNA 结构并调节 Cas13a 的 -切割活性，这丰富了 CRISPR-Cas13a 传感工具包，并将极大地扩展其应用范围。
