In Silico Design and Evaluation of CRISPR-Cas9 Guide RNAs Targeting the HTT Gene for Huntington's Disease Therapy

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disease caused by the expansion of CAG trinucleotides in the HTT gene, resulting in the formation of mutant huntingtin protein. This study focuses on the in silico design and analysis of CRISPR-Cas9 guide RNAs (gRNAs) directed to the HTT gene as a potential therapeutic intervention. The human HTT gene sequence (GRCh38) was downloaded from the NCBI database, and possible CRISPR target sites with the NGG protospacer adjacent motif (PAM) were located. Bioinformatics tools such as CHOPCHOP, CRISPOR, and Benchling were used to design candidate gRNAs. All gRNAs were assessed according to GC content, predicted on-target efficiency, and off-target potential. The content of GC was kept at the optimal level of 40-60%, and the efficiency scores were evaluated with the help of CFD-based prediction models. The off-target analysis was performed throughout the human genome with a maximum of three mismatches. Of the designed candidates, gRNA-2 had the highest predicted efficiency (0.81) and specificity with no observed off-target interactions, so it was the most promising candidate. This paper shows the utility of computational methods in determining the best gRNAs and forms the basis of additional experimental studies in the formulation of CRISPRbased therapies for Huntington's disease

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