A versatile CRISPR/Cas12a-based biosensing platform coupled with a target-protected transcription strategy

Xinyue Kang; Chao Lei; Jingjing Shi; Xiaoling Liu; Wei Ren; Chenghui Liu

doi:10.1016/j.bios.2022.114801

A versatile CRISPR/Cas12a-based biosensing platform coupled with a target-protected transcription strategy

Biosens Bioelectron. 2023 Jan 1:219:114801. doi: 10.1016/j.bios.2022.114801. Epub 2022 Oct 10.

Authors

Xinyue Kang¹, Chao Lei¹, Jingjing Shi¹, Xiaoling Liu², Wei Ren¹, Chenghui Liu³

Affiliations

¹ 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 Province, PR China.
² 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 Province, PR China. Electronic address: liuxl2018@snnu.edu.cn.
³ 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 Province, PR China. Electronic address: liuch@snnu.edu.cn.

PMID: 36270083
DOI: 10.1016/j.bios.2022.114801

Abstract

Besides the critical role in gene editing, CRISPR/Cas system also brings a new signal amplification mechanism to the development of next generation biosensing technologies. Herein, we have developed a versatile CRISPR/Cas12a sensing platform by combining a target protection-based transcription amplification strategy with the Cas12a-based signal amplification mechanism, which allows for the sensitive detection of both nucleic acid and non-nucleic acid targets. In this design, a rationally designed transcription template sequence is able to avoid Exonuclease I (Exo I) degradation only in the existence of the target-mediated binding events including either nucleic acid hybridization or protein-based affinity interactions. This target binding-induced protection effect can facilitate the subsequent transcription amplification to generate crRNA and activate the subsequent Cas12a trans-cleavage signal amplification mechanism to yield target dosage-responsive fluorescence signal. In contrast, if the target is absent, the protection-free transcription template will be completely digested by Exo I, thus no fluorescence response is produced. This new strategy well eliminates the T7 polymerase-associated non-specific transcription background and realizes the sensitive detection of various kinds of biomolecules including microRNA, protein, as well as exosome, broadening the application scenarios of CRISPR/Cas system in the field of bioanalysis and biosensing.

Keywords: CRISPR/Cas12a; Exosomes; Target protection; Transcription; microRNAs.