Harnessing enhanced CRISPR/Cas12a trans-cleavage activity with extended reporters and reductants for early diagnosis of Helicobacter pylori, the causative agent of peptic ulcers and stomach cancer

• Published in: Biosensors & bioelectronics 2023-02, Vol.222, p.114939-114939, Article 114939
• Main Authors: Habimana, Jean de Dieu, Mukama, Omar, Chen, Guiquan, Chen, Mengjun, Amissah, Obed Boadi, Wang, Lin, Liu, Yujie, Sun, Yirong, Li, Amy L., Deng, Sihao, Huang, Jufang, Yan, Xiao-xin, Rutaganda, Theobard, Mutangana, Dieudonne, Wu, Lin-Ping, Huang, Rongqi, Li, Zhiyuan
• Format: Article
• Language: English
• Subjects: Antigens, Bacterial - metabolism; Bacterial Proteins - genetics; Biosensing Techniques; Colorimetric readout; CRISPR-Cas Systems; Cytotoxins - genetics; Early Detection of Cancer; Enhanced LbCas12a; Extended reporters; Fluorescence readout; Genotype; Helicobacter Infections - diagnosis; Helicobacter Infections - genetics; Helicobacter Infections - metabolism; Helicobacter pylori; Helicobacter pylori - genetics; Helicobacter pylori - metabolism; Humans; Peptic Ulcer - diagnosis; Peptic Ulcer - genetics; Reducing Agents; Reductants; Stomach Neoplasms - diagnosis; Stomach Neoplasms - genetics
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2022.114939

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 13C 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. Schematic illustration of the CEXTRAR platform for real-time fluorescence and visual colorimetric monitoring of H. pylori. The clinical sample is first extracted using Triton X-100 (A), and subjected to LAMP amplification (B), followed by CRISPR detection using CEXTRAR (C) in one-pot reaction. The end-point result interpretation can be accomplished using real-time fluorescence, in-tube fluorescence, and lateral flow readouts (D). A more detailed mechanism of LAMP is described in (Notomi et al., 2015). [Display omitted]