Chen Siyu, Wang Rujia, Peng Shuang, Xie Shiyi, Lei Chunyang, Huang Yan, Nie Zhou
State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University Changsha 410082 P. R. China
Chem Sci. 2022 Jan 17;13(7):2011-2020. doi: 10.1039/d1sc05558e. eCollection 2022 Feb 16.
The CRISPR-Cas system has been repurposed as a powerful live-cell imaging tool, but its utility is limited to genomic loci and mRNA imaging in living cells. Here, we demonstrated the potential of the CRISPR-Cas system as a generalizable live-cell biosensing tool by extending its applicability to monitor diverse intracellular biomolecules. In this work, we engineered a CRISPR-Cas12a system with a generalized stimulus-responsive switch mechanism based on PAM-less conditional DNA substrates (pcDNAs). The pcDNAs with stimulus-responsiveness toward a trigger were constructed from the DNA substrates featuring no requirement of a protospacer-adjacent motif (PAM) and a bubble structure. With further leveraging the trans-cleavage activity of CRISPR-Cas12a for signal reporting, we established a versatile CRISPR-based live-cell biosensing system. This system enabled the sensitive sensing of various intracellular biomolecules, such as telomerase, ATP, and microRNA-21, making it a helpful tool for basic biochemical research and disease diagnostics.
CRISPR-Cas系统已被改造成为一种强大的活细胞成像工具，但其应用仅限于活细胞中的基因组位点和mRNA成像。在这里，我们通过将CRISPR-Cas系统的适用性扩展到监测多种细胞内生物分子，证明了其作为一种通用的活细胞生物传感工具的潜力。在这项工作中，我们基于无PAM的条件性DNA底物（pcDNA）设计了一种具有通用刺激响应开关机制的CRISPR-Cas12a系统。对触发因素具有刺激响应性的pcDNA是由不需要原间隔相邻基序（PAM）和气泡结构的DNA底物构建而成。通过进一步利用CRISPR-Cas12a的反式切割活性进行信号报告，我们建立了一个通用的基于CRISPR的活细胞生物传感系统。该系统能够灵敏地检测各种细胞内生物分子，如端粒酶、ATP和微小RNA-21，使其成为基础生化研究和疾病诊断的有用工具。
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