Exploration of new ways for CRISPR/Cas12a activation: DNA hairpins without PAM and toehold and single strands containing DNA and RNA bases

IF 4.1 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Wen He , Xinyu Li , Xinmin Li , Minghui Guo , Mengxuan Zhang , Ruiwei Hu , Menghan Li , Shijia Ding , Yurong Yan
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Abstract

The CRISPR/Cas12a system is emerging as a promising candidate for next-generation diagnostic biosensing platforms, with the discovery of new activation modes greatly expanding its applications. Here, we have identified two novel CRISPR/Cas12a system activation modes: PAM- and toehold-free DNA hairpins, and DNA-RNA hybrid strands. Utilizing a well-established real-time fluorescence method, we have demonstrated a strong correlation between DNA hairpin structures and Cas12a activation. Compared with previously reported activation modes involving single-stranded DNA and PAM-contained double-stranded DNA, the DNA hairpin activation way exhibits similar specificity and generality. Moreover, our findings indicate that increasing the number of RNA bases in DNA-RNA hybrid strands can decelerate the kinetics of Cas12a-triggered trans-cleavage of reporter probes. These newly discovered CRISPR/Cas12a activation ways hold significant potential for the development of high-performance biosensing strategies.

探索 CRISPR/Cas12a 激活的新途径:不含 PAM 和趾扣的 DNA 发夹以及含有 DNA 和 RNA 碱基的单链。
CRISPR/Cas12a 系统正在成为下一代诊断生物传感平台的理想候选系统,新激活模式的发现极大地扩展了它的应用范围。在这里,我们发现了两种新的 CRISPR/Cas12a 系统激活模式:PAM和无趾hold DNA发夹以及DNA-RNA杂交链。利用成熟的实时荧光方法,我们证明了 DNA 发夹结构与 Cas12a 激活之间的密切联系。与之前报道的涉及单链 DNA 和含有 PAM 的双链 DNA 的激活方式相比,DNA 发夹激活方式具有相似的特异性和通用性。此外,我们的研究结果表明,增加DNA-RNA杂交链中的RNA碱基数量可以减慢Cas12a触发的报告探针反式裂解的动力学。这些新发现的CRISPR/Cas12a激活方式为开发高性能生物传感策略提供了巨大潜力。
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来源期刊
Journal of biotechnology
Journal of biotechnology 工程技术-生物工程与应用微生物
CiteScore
8.90
自引率
2.40%
发文量
190
审稿时长
45 days
期刊介绍: The Journal of Biotechnology has an open access mirror journal, the Journal of Biotechnology: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. The Journal provides a medium for the rapid publication of both full-length articles and short communications on novel and innovative aspects of biotechnology. The Journal will accept papers ranging from genetic or molecular biological positions to those covering biochemical, chemical or bioprocess engineering aspects as well as computer application of new software concepts, provided that in each case the material is directly relevant to biotechnological systems. Papers presenting information of a multidisciplinary nature that would not be suitable for publication in a journal devoted to a single discipline, are particularly welcome.
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