دورية أكاديمية
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.
| العنوان: | 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. |
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| المؤلفون: | Habimana JD; 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., Mukama O; 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., Chen G; Department of Gastroenterology, Affiliated Dongguan Hospital, Southern Medical University-Dongguan People's Hospital, Dongguan, 523059, China., Chen M; Department of Gastroenterology, Affiliated Dongguan Hospital, Southern Medical University-Dongguan People's Hospital, Dongguan, 523059, China., Amissah OB; 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., Wang L; 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; Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China., Liu Y; 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., Sun Y; 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., Li AL; 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., Deng S; Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha, China., Huang J; Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha, China., Yan XX; Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha, China., Rutaganda T; College of Science 205 Mugar Life Sciences, Northeastern University, 360 Huntington Avenue Boston, MA, 02115, USA., Mutangana D; Department of Biology, College of Science and Technology, University of Rwanda, Avenue de l'armée, Kigali, P.O. Box: 3900, Rwanda., Wu LP; 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., Huang R; 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; Guangzhou Qiyuan Biomedical Co., Ltd, Guangzhou, China. Electronic address: huang_rongqi@gibh.ac.cn., Li Z; 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 Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha, China; Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China; GZMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou, China; GIBH-HKU Guangdong-HongKong Stem Cell and Regenerative Medicine Research Centre, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, Guangzhou, China; Guangzhou Qiyuan Biomedical Co., Ltd, Guangzhou, China. Electronic address: li_zhiyuan@gibh.ac.cn. |
| المصدر: | Biosensors & bioelectronics [Biosens Bioelectron] 2023 Feb 15; Vol. 222, pp. 114939. Date of Electronic Publication: 2022 Nov 20. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Advanced Technology Country of Publication: England NLM ID: 9001289 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-4235 (Electronic) Linking ISSN: 09565663 NLM ISO Abbreviation: Biosens Bioelectron Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Oxford : Elsevier Advanced Technology Original Publication: [Barking, Essex, England] : Elsevier Applied Science, 1989- |
| مواضيع طبية MeSH: | Helicobacter pylori*/genetics , Helicobacter pylori*/metabolism , Stomach Neoplasms*/diagnosis , Stomach Neoplasms*/genetics , Biosensing Techniques* , Peptic Ulcer*/diagnosis , Peptic Ulcer*/genetics , Helicobacter Infections*/diagnosis , Helicobacter Infections*/genetics , Helicobacter Infections*/metabolism, Humans ; Antigens, Bacterial/metabolism ; Bacterial Proteins/genetics ; Reducing Agents ; CRISPR-Cas Systems ; Early Detection of Cancer ; Genotype ; Cytotoxins/genetics |
| مستخلص: | 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 13 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. Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2022 Elsevier B.V. All rights reserved.) |
| فهرسة مساهمة: | Keywords: Colorimetric readout; Enhanced LbCas12a; Extended reporters; Fluorescence readout; Helicobacter pylori; Reductants |
| المشرفين على المادة: | 0 (Antigens, Bacterial) 0 (Bacterial Proteins) 0 (Reducing Agents) 0 (Cytotoxins) |
| تواريخ الأحداث: | Date Created: 20221202 Date Completed: 20221227 Latest Revision: 20221230 |
| رمز التحديث: | 20231215 |
| DOI: | 10.1016/j.bios.2022.114939 |
| PMID: | 36459819 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1873-4235 |
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| DOI: | 10.1016/j.bios.2022.114939 |