Gene Editing / Rare Genetic Disorders
Project 1: Designing Novel Therapeutic Strategies for Rare
Genetic Diseases using CRISPR-Based
Project Description:
This project presents a computational CRISPR-Cas9 functional genomics pipeline to design and evaluate therapeutic strategies for Cystic Fibrosis (CF) — a rare geneticdisordercausedbymutationsintheCFTRgene. The project integrates sgRNA design, functional genomic analysis, and gene ontology (GO) enrichment toexplorehowgeneeditingmightrestoreCFTRfunction ortargetitsregulatorynetworkfortherapy.
Advanced Methodology Pipeline:
1. Data Preprocessing
•Input: Pathogenic CFTR variants were obtainedfromtheClinVarVCFfile.
•Filtering was applied to retain only pathogenicvariantslinkedtoCF.
•Mutationdatawasformattedforinputinto sgRNAdesigntools.
2. sgRNA Design using CRISPOR
•UsedCRISPORtodesignhigh-efficiency, high-specificity sgRNAs for the identified CFTRmutations.
•sgRNAs were filtered using Moreno-Mateosetal.thresholds.
Key evaluation parameters
•Efficiencyscores
•Specificityscores
•Off-targetpotential(insilico)
3. Functional Genomics & Frameshift Analysis
Simulatedindel mutationsinducedby sgRNAsandcalculated:
•Frameshiftprobabili
•tyIndelefficiency
Usedmatplotlibto visualizeefficiency distributionsand mutationprofiles
Figure 1: Histogram or violin plot of indel efficiency and frameshift likelihood across all sgRNAs
4. Gene Ontology & Pathway Enrichment
•sgRNA-associated gene targets were analyzedusing:
1.DAVID
2.Enrichr(GO:BP,CC,MF)
3.KEGGpathwaymapsforCFTR
•Common enriched term: GO:0030163 –Proteincatabolicprocess
Figure 2: Word cloud of biological processes
5. Network & Functional Analysis
•Identified candidate genes related to CFTR degradation, folding, and stress response:
⚬ STUB1,SQSTM1,UCHL1,PRKN
•Generated a GO term interaction network using Cytoscape to visualize biological interconnections.
Figure 4: GO term network map with clustered biological pathways
6. Molecular Simulation & Therapeutic Insights
•Though sgRNAs did not directly overlap with top enriched GO terms, two molecularstrategieswereproposed:
a. Docking sgRNAs to CFTR-related regulatoryelements
Protein-ligandinteractionmodelingformutant
CFTR with small molecules to explore CRISPRsynergy
Project Outcomes:
Designedandvalidated multiplesgRNAs targetingpathogenic CFTRmutations.
Identifiedkeybiological processes(e.g.,protein catabolism,ubiquitin pathways)contributingto CFTRdysfunction.
Providedareproduciblein silicopipelinethatconnects variantannotation→ sgRNAdesign→gene networkanalysis.
Conclusion:
ThisprojectbridgesCRISPRsgRNAdesignandfunctional interpretationthroughcomputationalmodelingforraregenetic diseases,usingCFTRasamodelgene.Althoughnodirect sgRNA-GOlinkwasfound,theenrichmentanalysisandgene prioritizationpresentstrongleadsforpreclinicalCRISPR-based therapeuticdevelopment.
Key Contributions:
Developed a therapeutic design pipeline ready for lab validation.
Identified functionally significant genes for indirect CRISPR targeting.
Proposed molecular simulation strategies to explore CRISPR-drug co-therapies.
