In Silico Design and Evaluation of CRISPR Cytosine Base Editing Strategy for Potential Correction of the ASPA Variant Associated with Canavan Disease

Abstract

Background Canavan disease is a rare autosomal recessive neurodegenerative leukodystrophy caused by pathogenic variants in the ASPA gene, for which effective molecularly targeted therapies remain limited. The NM_000049.4:c.691T>C (p.Tyr231His) variant introduces a pathogenic C•G base pair that is potentially amenable to correction using cytosine base editing (CBE). This study aimed to design and computationally evaluate a CBE-based CRISPR strategy for potential correction of this ASPA variant. Methods An integrated in silico workflow was used to characterize the ASPA p.Tyr231His variant and design a CBE-compatible sgRNA. Variant pathogenicity, conservation, and structural stability were assessed using multiple computational tools. Candidate sgRNAs were designed and evaluated for editing-window positioning, PAM compatibility, specificity, GC content, bystander editing, and secondary structure. Genome-wide off-target potential was further assessed using CRISPRitz against the GRCh38 reference genome. Results The ASPA p.Tyr231His variant was exceptionally rare (gnomAD allele frequency: 1.86 × 10−6), highly conserved, and consistently predicted to be deleterious, with DynaMut2 indicating reduced protein stability (ΔΔG = −2.04 kcal/mol). Of two candidate sgRNAs, one was selected based on optimal editing-window positioning, absence of predicted bystander editing, favourable GC content (45%), and specificity scores (MIT: 82; CFD: 93). RNAfold predicted minimal secondary-structure constraints (MFE: 0.00 kcal/mol). CRISPRitz identified 108 potential off-target sites, with none containing fewer than three mismatches, no bulge-associated sites, and only three predicted sites within exonic regions. Conclusions The integrated computational analysis identified a candidate sgRNA with favourable predicted editing characteristics and genome-wide specificity for potential CBE-mediated correction of the ASPA c.691T>C (p.Tyr231His) variant. These findings provide a rational framework for prioritizing this guide for experimental evaluation. Further cell-based and in vivo studies are required to establish editing efficiency, editing purity, bystander activity, off-target effects, and restoration of ASPA function before therapeutic applicability can be assessed.