Design and optimization of gene editing-based gRNA for detection of Vibrio cholerae O1

Subject Areas : Microbial Biotechnology

Mina Khoddamy Vayghan ¹ , Mehdi Zeinoddini ^{2 *} , Zahra AleAgha ³ , Alireza Saeedinia ⁴

1 - Department of Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
2 - 2Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Iran;
3 - Department of Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran.
4 -

Received: 2025-03-02 Accepted : 2025-05-25 Published : 2025-06-10

Keywords: Guide RNA, Vibrio cholerae, CRISPR, Molecular diagnosis.,

Abstract :

Background & Objectives: Gene editing technology, especially the CRISPR-Cas12a system, has emerged as a novel method for rapid and accurate pathogen detection. This study aims to design guide RNAs for identifying three main genes of Vibrio cholerae O1: CtxA, CtxB, and OmpW.

Materials & Methods: The target gene sequences were extracted in FASTA format and guide RNAs were designed using three online tools: Chop chop, Crispor, and Cas designer. Parameters such as Cas protein type, PAM sequence, number of target sites, organism specificity, and sequence structural features were considered. After optimization, sequences were prioritized based on functionality, secondary structure, GC content, and sense or antisense orientation. Design accuracy was confirmed by comparing software outputs and analyzing secondary structures using RNAfold and GC Calculator. The best sequences for each target gene were selected and expression vectors containing corresponding cassettes were constructed.

Results: Optimized guide RNAs, considering the specific characteristics of the CRISPR-Cas12a system, demonstrated the ability to accurately and specifically identify the three target genes of Vibrio cholerae in silico. These designs were structurally stable and exhibited an appropriate GC content, which can ensure optimal performance in CRISPR-Cas12a-based diagnostic systems. Furthermore, comparison of software results and structural analyses confirmed the validity and reliability of these designs.

Conclusion: The CRISPR-Cas12a-based diagnostic method developed here offers a rapid, accurate, and cost-effective approach for Vibrio cholerae detection and can contribute to controlling and preventing cholera outbreaks.

References:


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