Identification of CRISPR/Cas12a (Cpf1) guideRNA Sequence Targeting the Mitochondrial DNA D-loop Region in Wild Pig (Sus scrofa) Through Homology Difference and Mismatch Analysis
DOI:
https://doi.org/10.48048/tis.2024.7603Keywords:
Benchling, gRNA CRISPR/Cas12a, Biosensor, mtDNA D-loop, Wild pig (Sus scrofa), In silicoAbstract
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) or CRISPR-associated (Cas) System has become a major gene editing tool. Gene editing with CRISPR requires the Cas protein and the corresponding guide RNA (gRNA). However, low cleavage efficiency and off-target effects can impede the application of the CRISPR/Cas system. Therefore, the determination of specific gRNAs is essential. In biosensor applications, CRISPR/Cas12a can enhance specificity and sensitivity in identifying target genes due to the trans-cleavage activity of Cas12a (Cpf1). The mtDNA D-loop sequence is the most variable part of mtDNA, making it suitable for distinguishing between species. Consequently, the objective of this study was to determine the gRNA sequence of the D-loop of wild pig mtDNA in silico. Candidate gRNAs were predicted using the Benchling application with the assistance of the GenBank database. The gRNA candidates were subsequently subjected to a homology difference analysis using BLAST nucleotide and a mismatch test using Jalview. Among several candidates, candidate 1 was selected as the best option, with an Off-target value of 99.8. The homology difference analysis against competitors and the mismatch test against the Sus genus resulted in high E-values and high percentage values, respectively. This suggests that the candidate will not recognize other species but can detect members of the Sus scrofa species. These gRNA candidates can be selectively and sensitively applied to biosensors for the detection of meat adulteration.
HIGHLIGHTS
- The D-loop exhibits high variability, make it useful for distinguishing between species
- gRNA as an enabler of CRISPR/Cas12a to identify the target and initiate cleavage activity
- The candidate 1 was selected with the sequence 5'-GAT TGT CGT GCC GGA TCA TGA GTT-3'
- The application of this strategy is aimed at determining the halal status of food products
- The application will encompass both quantitative and qualitative aspects
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