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 , علی رضا سعیدی نیا 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:


1. Arianzad SA, Zeinoddini M, Haddadi A, Nazarian S, Sajedi RH. The effect of recombinant IpaB protein on the development of immune responses against Shigella dysenteriae in Guinea Pig. J Microb World.2022;15(3):170-182. )In Persian(
2. Zeinoddini M, Saeedinia AR, Sadeghi V. Rapid detection of Vibrio cholerae using hexaplex PCR assay. J Police Med. 2014;3(2):77-84. )In Persian(
3. Rossetti M, Gamberini D, Sforza S, Corradini R. Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters. Nucleic Acids Res. 2022;50(14):8377-8391.
4. Su W, Li J, Yang Y, Wang G, Yu Z, Zeng F. CRISPR/Cas systems for the detection of nucleic acid and non-nucleic acid targets. Nano Res. 2023;16(7):9940-9953.
5. van Dongen JE, Berendsen JTW, Steenbergen RDM, Wolthuis RMF, Eijkel JCT, Segerink LI. Point-of-care CRISPR/Cas nucleic acid detection: recent advances, challenges and opportunities. Biosens Bioelectron. 2020;166:112445.
6. Saito M, Xu P, Faure G, Maguire S, Kannan S, Altae-Tran H. Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature. 2023;620(7974):660-668.
7. Huang T, Zhang R, Li J. CRISPR-Cas-based techniques for pathogen detection: retrospect, recent advances, and future perspectives. J Adv Res. 2023;50:69-82.
8. Qiu M, Zhou XM, Liu L. Improved strategies for CRISPR-Cas12-based nucleic acids detection. J Anal Test. 2022;6(1):44-52.
9. Jafari H, Ranjbari M. Alkaloids derived from marine algae and their therapeutic properties – mini-review. J Mar Med. 2019;1(1):3-10. )In Persian(
10. Rananaware SR, Sahu P, Sharma A, Kumar B. Programmable RNA detection with CRISPR-Cas12a. Nat Commun. 2023;14(1):5409.
11. Kolahdouzmohammadi M, Pahlavan S, Tahamtani Y, Sotoodehnejadnematalahi F, Totonchi M. Step by step design of popular and common vectors in genetic manipulation using CRISPR/Cas9 system. HBI Journals. 2022;27(4):93-109. )In Persian(
12. Kim S, Ji S, Koh HR. CRISPR as a diagnostic tool. Biomolecules. 2021;11(8):1162.
13. Zhang J, Nielsen R, Wei Q. Primer design and PCR optimization in molecular biology research. Methods Mol Biol. 2021;2233:27-45.
14. Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 2019;47(W1):W171-W174.
15. Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018;46(W1):W242-W245.
16. Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL. The Vienna RNA websuite. Nucleic Acids Res. 2008;36(Web Server issue):W70-W74.
17. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827-832.
18. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308.
19. Lu P, Wang Y, Wang S, Li Q, Huang S, Ma J. Visual identification and serotyping of toxigenic Vibrio cholerae serogroups O1 and O139 with CARID. Front Cell Infect Microbiol. 2022;12:863435.
20. Liang Q, Tu B, Cui L. Recombinant T7 RNA polymerase production using ClearColi BL21(DE3) and animal-free media for in vitro transcription. Appl Microbiol Biotechnol. 2024;108(1):41.
21. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759-771.
22. Bandyopadhyay A, Rai A, Rai A, Chanda D, Mishra AK, Tiwari S. CRISPR-Cas12a (Cpf1): a versatile tool in the plant genome editing tool box for agricultural advancement. Front Plant Sci. 2020;11.
23. Baldanta S, Guevara G, Navarro-Llorens JM. SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria. Microb Cell Fact. 2022;21(1):103.
24. Wang H, Zhou J, Lei J, Mo G, Wu Y, Liu H. Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system. PLoS Biol. 2024;22(5):e3002619.
25. Gao Z, Herrera-Carrillo E, Berkhout B. A single H1 promoter can drive both guide RNA and endonuclease expression in the CRISPR-Cas9 system. Mol Ther Nucleic Acids. 2019;14:32-40.
26. Calvopina-Chavez DG, Gardner MA, Griffitts JS. Engineering efficient termination of bacteriophage T7 RNA polymerase transcrption. G3 (Bethesda). 2022;12(6).
27. Yu L, Marchisio MA. Scaffold RNA engineering in type V CRISPR-Cas systems: a potent way to enhance gene expression in the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 2024;52(3):1483-1497.
28. Bigelyte G, Duchovska B, Zedaveinyte R, Dapkute D, Gasiunas G. Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing. Nucleic Acids Res. 2024;52(6):3234-3248.
29. Bains JK, Bhaduri S, Nischal K, Jain P. Cell-free transcription-translation system: a dual read-out assay to characterize riboswitch function. Nucleic Acids Res. 2023;51(15):e82.
30. Kim H, Lee WJ, Oh Y, Kang SH, Hur JK, Lee H. Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide. Nucleic Acids Res. 2020;48(15):8601-8616.
31. Mardashti Z, Zeinoddini M, Saeedi Nia AR. Design of guide RNA for detection of Salmonella typhimurium based on CRISPR-Cas12. Biotechnology.) In press, Persian(