Engineered FnCas9 mediated mutation profiling for clarithromycin resistance in Helicobacter pylori strains isolated from Indian patients with gastrointestinal disorders

• Published in: Microchemical journal 2024-12, Vol.207, p.112051, Article 112051
• Main Authors: Chakraborty, Shraddha, Rana, Shubham, Gulati, Sneha, Chaudhary, Shubham, Panigrahi, Manas K., Hallur, Vinay K., Maiti, Souvik, Chakraborty, Debojyoti, Makharia, Govind K.
• Format: Article
• Language: English
• Subjects: 23S ribosomal DNA; Antibiotic resistance; CRISPR-Cas9; FELUDA; Gastrointestinal diseases; Helicobacter pylori
• ISSN: 0026-265X
• DOI: 10.1016/j.microc.2024.112051

[Display omitted] •Clarithromycin resistance in H. pylori strains, due to 23S rDNA mutations, complicates eradication efforts in developing countries.•We hereby introduce an engineered FnCas9-based assay to detect clarithromycin resistance in H. pylori strains isolated from patient biopsies.•Outcome of in vitro cleavage studies and membrane-based lateral flow tests (FELUDA) perfectly align with Sanger sequencing and RFLP.•Highlights the potential of this novel CRISPR-based diagnostic methodology to be developed as a point-of-care mutation detection strategy. Helicobacter pylori is a highly prevalent gut pathogen with reported implications in a wide range of gastrointestinal disorders. Antibiotic based therapy, especially with clarithromycin is one of most effective treatment strategies against H. pylori. However, rising global prevalence of clarithromycin resistance in certain H. pylori strains, primarily attributed to point mutations in the 23S ribosomal RNA coding gene, pose a major challenge in effective eradication of this pathogen. There are a number of established methodologies devised for H. pylori mutation detection, so as to provide a tailored treatment plan to the patients and resist further transmissions of antibiotic resistant strains. However, there is no ‘gold standard’ method available to detect mutation status in clinical isolates of H. pylori from infected patients. CRISPR-Cas9 based technologies have revolutionized the field of mutation detection in biological samples, particularly during the recent COVID-19 pandemic. Although multiple assays have been reported for detection of H. pylori in clinical samples including CRISPR diagnostics (CRISPRDx) platforms, there is no such assay reported till date to detect specific mutations that confer antibiotic resistance to this pathogen. In this study, we have developed an assay using engineered FnCas9 (en31-FnCas9) protein to effectively detect the A2142G and A2143G mutations in the 23S rDNA of H. pylori strains isolated from gastric biopsy samples. The data from in vitro cleavage assays and strip-based lateral flow tests using en31-FnCas9 and guide RNAs targeting the conserved and mutated loci of H. pylori 23S rDNA are in perfect congruence with the data from Sanger sequencing and restriction fragment length polymorphism (RFLP) analysis. Our results indicate that en31-FnCas9 based mutation analysis can be deployed as an efficient diagnostic methodology to detect clarithromycin susceptibility in p