影响通用光控CRISPR诊断中Cas12a crRNA活性的关键核苷酸的鉴定

IF 15.7 1区 综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES
Tian Tian, Hongrui Xiao, Xinyi Guo, Yuxin Chen, Zhiqiang Qiu, Ting Zhang, Meiyu Chen, Weiwei Qi, Peige Cai, Meng Cheng, Xiaoming Zhou
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引用次数: 0

摘要

开发一锅检测是增强基于crispr的分子诊断适用性的关键策略;然而,由于CRISPR切割干扰核酸扩增模板,阻碍了这一过程。光调节策略提供了一种理想的解决方案来抑制不希望的CRISPR切割,同时保持检测效率。然而,现有的光控CRISPR诊断方法在通用性、成本和检测效率等方面存在局限性。在这项研究中,我们系统地研究了重复识别序列(RRS)突变对切割活性的影响,重复识别序列(RRS)是Cas12a crRNA直接重复(DR)区域内的一个四核苷酸片段。我们观察到位置3或4的突变几乎消除了crRNA的活性。基于这一发现,我们在3号和4号位置引入了6-硝基胡椒酰氧基甲基(NPOM)光笼修饰。4号位置的光笼可以最有效地抑制酶活性和最佳的光介导激活。我们利用这一发现开发了一种光控CRISPR诊断方法,实现了一种普遍适用的一锅检测策略。此外,通过结合crRNA夹板策略,这种预先准备的试剂可以适用于几乎任何靶基因的检测。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics

Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics

Developing a one-pot assay is a critical strategy for enhancing the applicability of CRISPR-based molecular diagnostics; however, it is hindered by CRISPR cleavage interfering with nucleic acid amplification templates. Photo-regulation strategies provide an ideal solution to suppress undesired CRISPR cleavage while maintaining detection efficiency. However, existing photo-controlled CRISPR diagnostic methods face limitations in universality, cost, and detection efficiency. In this study, we systematically examine the impact of mutations in the repeat recognition sequence (RRS), a four-nucleotide segment within the Cas12a crRNA direct repeat (DR) region, on cleavage activity. We observe that mutations at positions 3 or 4 nearly abolished crRNA activity. Based on this discovery, we introduce 6-nitropiperonyloxymethyl (NPOM) photo-caging modifications at positions 3 and 4. Photo-caging at position 4 demonstrates the most effective suppression of enzymatic activity and optimal light-mediated activation. We leverage this finding to develop a photo-controlled CRISPR diagnostic method, enabling a universally adaptable one-pot detection strategy. Furthermore, by incorporating a crRNA splinting strategy, this pre-preparable reagent can be adapted for the detection of virtually any target gene.

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来源期刊
Nature Communications
Nature Communications Biological Science Disciplines-
CiteScore
24.90
自引率
2.40%
发文量
6928
审稿时长
3.7 months
期刊介绍: Nature Communications, an open-access journal, publishes high-quality research spanning all areas of the natural sciences. Papers featured in the journal showcase significant advances relevant to specialists in each respective field. With a 2-year impact factor of 16.6 (2022) and a median time of 8 days from submission to the first editorial decision, Nature Communications is committed to rapid dissemination of research findings. As a multidisciplinary journal, it welcomes contributions from biological, health, physical, chemical, Earth, social, mathematical, applied, and engineering sciences, aiming to highlight important breakthroughs within each domain.
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