CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing, 100049, China.
CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing, 100049, China; Department of Biology, College of Science and Technology, University of Rwanda, Avenue de l'armée, Kigali, P.O. Box: 3900, Rwanda.
Biosens Bioelectron. 2023 Feb 15;222:114939. doi: 10.1016/j.bios.2022.114939. Epub 2022 Nov 20.
Developing rapid and non-invasive diagnostics for Helicobacter pylori (HP) is imperative to prevent associated diseases such as stomach gastritis, ulcers, and cancers. Owing to HP strain heterogeneity, not all HP-infected individuals incur side effects. Cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) genes predominantly drive HP pathogenicity. Therefore, diagnosing CagA and VacA genotypes could alert active infection and decide suitable therapeutics. We report an enhanced LbCas12a trans-cleavage activity with extended reporters and reductants (CEXTRAR) for early detection of HP. We demonstrate that extended ssDNA reporter acts as an excellent signal amplifier, making it a potential alternative substrate for LbCas12a collateral activity. Through a systematic investigation of various buffer components, we demonstrate that reductants improve LbCas12a trans-cleavage activity. Overall, our novel reporter and optimal buffer increased the trans-cleavage activity to an order of 16-fold, achieving picomolar sensitivity (171 pM) without target pre-amplification. Integrated with loop-mediated isothermal amplification (LAMP), CEXTRAR successfully attained attomolar sensitivity for HP detection using real-time fluorescence (43 and 96 aM), in-tube fluorescence readouts (430 and 960 aM), and lateral flow (4.3 and 9.6 aM) for CagA and VacA, respectively. We also demonstrate a rapid 2-min Triton X-100 lysis for clinical sample analysis, which could provide clinicians with actionable information for rapid diagnosis. CEXTRAR could potentially spot the C urea breath test false-negatives. For the first time, our study unveils an experimental outlook to manipulate reporters and reconsider precise cysteine substitution via protein engineering for Cas variants with enhanced catalytic activities for use in diagnostics and genetic engineering.
开发快速、非侵入性的幽门螺杆菌（HP）诊断方法对于预防相关疾病（如胃炎、溃疡和癌症）至关重要。由于 HP 菌株的异质性，并非所有感染 HP 的个体都会产生副作用。细胞毒素相关基因 A（CagA）和空泡细胞毒素 A（VacA）基因主要驱动 HP 的致病性。因此，诊断 CagA 和 VacA 基因型可以提示是否存在活动性感染，并决定合适的治疗方法。我们报告了一种增强的 LbCas12a 跨切割活性，带有扩展报告子和还原剂（CEXTRAR），用于早期检测 HP。我们证明，扩展的 ssDNA 报告子可作为出色的信号放大器，使其成为 LbCas12a 旁侧活性的潜在替代底物。通过对各种缓冲成分的系统研究，我们证明还原剂可提高 LbCas12a 的跨切割活性。总体而言，我们的新型报告子和最佳缓冲液将跨切割活性提高了 16 倍，在没有靶标预扩增的情况下达到皮摩尔灵敏度（171 pM）。与环介导等温扩增（LAMP）集成，CEXTRAR 成功地实现了使用实时荧光（43 和 96 aM）、管内荧光读数（430 和 960 aM）和侧流（4.3 和 9.6 aM）对 CagA 和 VacA 的 attomolar 灵敏度检测。我们还展示了一种快速的 2 分钟 Triton X-100 裂解用于临床样本分析，这可以为临床医生提供快速诊断的决策信息。CEXTRAR 可能会发现 C 尿素呼气试验的假阴性。这是首次通过操纵报告子并通过蛋白工程重新考虑精确半胱氨酸取代，为具有增强催化活性的 Cas 变体用于诊断和基因工程提供了实验前景。
