State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, People's Republic of China.
School of Medical Laboratory, College of Medical Technology, Tianjin Medical University, Guangdong Road, Tianjin 300203, People's Republic of China.
Anal Chem. 2022 Jun 7;94(22):8050-8057. doi: 10.1021/acs.analchem.2c01401. Epub 2022 May 26.
The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing applications. However, the lack of exploration on the fundamental properties of CRISPR/Cas12a not only discourages further in-depth studies of the CRISPR/Cas12a system but also limits the design space of CRISPR/Cas12a-based applications. Herein, a "RESET" effect (random extending sequences enhance trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, a single-stranded DNA, which is too short to work as the activator, can efficiently activate CRISPR/Cas12a after being extended a random sequence from its 3'-end, even when the random sequence folds into secondary structures. The finding of the "RESET" effect enriches the CRISPR/Cas12a-based sensing strategies. Based on this effect, two CRISPR/Cas12a-based biosensors are designed for the sensitive and specific detection of two biologically important enzymes.
CRISPR/Cas12a 的转切割活性已被广泛应用于生物传感应用。然而，对 CRISPR/Cas12a 的基本性质缺乏探索，不仅阻碍了对 CRISPR/Cas12a 系统的进一步深入研究，也限制了基于 CRISPR/Cas12a 的应用的设计空间。在此，发现了 CRISPR/Cas12a 转切割活性的“重置”效应（随机延伸序列增强转切割活性）。也就是说，单链 DNA 太短，不能作为激活剂，但当其 3'-端延伸一个随机序列后，即使该随机序列折叠成二级结构，也能有效地激活 CRISPR/Cas12a。“重置”效应的发现丰富了基于 CRISPR/Cas12a 的传感策略。基于这一效应，设计了两种基于 CRISPR/Cas12a 的生物传感器，用于灵敏和特异性检测两种重要的生物酶。
