Diseño IN SILICO de un sistema CRISPR interferencia dirigido a inhibir la expresión del oncogén HOXB13 en cáncer de próstata

Abstract

Prostate cancer constitutes one of the major malignant neoplasms in the male population and presents a relevant fraction of cases with a hereditary component. Among the genes associated with this type of cancer, HOXB13 has been identified as a central regulator in prostate tumor progression, with critical transcriptional and epigenetic functions. However, its nature as a transcription factor classifies it as a pharmacologically non-druggable target, limiting conventional therapeutic strategies. In this context, CRISPR-mediated interference (CRISPRi) emerges as an alternative for specific transcriptional silencing without inducing DNA double-strand breaks. The objective of this research was to design in silico a CRISPRi system aimed at inhibiting the expression of the HOXB13 oncogene in prostate cancer. To this end, a computational methodological workflow structured in three phases was developed. In Phase 1, the HOXB13 genomic locus was characterized in the human genome (GRCh38/hg38), and a regulatory region proximal to the transcription start site was delimited. In Phase 2, guide RNAs were designed and evaluated using the CHOPCHOP, CRISPOR, Cas-OFFinder, and RNAfold platforms, integrating criteria of relative position to the TSS, genomic specificity, exhaustive off-target analysis, and guide RNA structural consistency. In Phase 3, in silico design of CRISPRi plasmid constructs derived from a validated lentiviral vector was performed, incorporating the selected guides without modifying the backbone architecture. The results allowed the prioritization of five crRNAs with profiles compatible with transcriptional interference, absence of critical off-targets, and functional secondary structures. Exploratory statistical analysis indicated a homogeneous set among candidates, without marked global differences, which justified the hierarchical integration of multiple parameters for rational prioritization. The final plasmid design coherently integrates the hU6–sgRNA cassette and the dCas9-KRAB effector, constituting a consistent platform for subsequent studies. Overall, this work establishes a reproducible computational design of a CRISPRi system targeting HOXB13, laying the methodological foundations for future experimental validation in prostate cancer models.