Workshop Structure

Module 1: CRISPR Basics (What You Need Before Editing)

• CRISPR history and why Cas9 became a game-changer.
• Key components: gRNA, PAM, Cas9 nuclease, target DNA.
• How CRISPR finds and cuts DNA (simple, step-by-step explanation).
• CRISPR vs older genome editing tools (TALENs, ZFNs) overview.

Module 2: Designing a CRISPR Editing Strategy

• Defining the biological goal: knockout, knock-in, correction, tagging.
• Choosing target regions: exons, functional domains, isoforms, splice variants.
• Editing outcomes: what happens after the cut and why results vary.
• Project planning checklist: what must be decided before ordering reagents.

Module 3: gRNA Design Principles (Potency + Specificity)

• What makes a good gRNA: sequence features and PAM constraints.
• Off-target risks: mismatches, seed region, repetitive sequences.
• Ranking gRNAs: balancing on-target efficiency and specificity.
• Hands-on workflow: designing 3–5 gRNAs for a chosen gene (tool-based approach).

Module 4: Repair Pathways (NHEJ vs HDR) and Editing Outcomes

• NHEJ for gene knockout: indels and frameshift logic.
• HDR for precise edits: donor design basics (ssODN/plasmid donors concept).
• Improving HDR efficiency: timing, cell cycle, and experimental strategies (overview).
• Common outcome issues: mosaicism, partial edits, unexpected indels.

Module 5: Delivery Methods & Experimental Setup

• Delivery options: plasmid, mRNA, RNP (riboprotein complex) overview.
• Transfection/transduction basics (conceptual): cells, reagents, and optimization.
• Controls that matter: non-targeting controls, positive controls, mock control.
• Safety basics: handling, containment, and responsible lab practices.

Module 6: Validation & Quality Control (Proving Your Edit Worked)

• Genotyping strategy: PCR design, amplicon sizing, sequencing options.
• T7E1/Surveyor assays (concept) and when sequencing is better.
• Protein-level validation: Western blot/functional assays overview.
• Interpreting results and troubleshooting weak or mixed edits.

Module 7: Applications in Research and Biotechnology

• Functional genomics: gene function discovery and pathway mapping.
• Disease models: cell lines, organoids, and animal models (overview).
• Biotech applications: agriculture, industrial biology, diagnostics (overview).
• Clinical frontier overview: gene therapy basics and safety concerns.

Module 8: Ethics, Biosafety & Responsible Genome Editing

• Ethical boundaries: somatic vs germline editing and why it matters.
• Risk awareness: off-target effects, unintended consequences, and misuse concerns.
• Data integrity: documentation, transparency, and reproducible reporting.
• Responsible communication: avoiding overclaiming and hype.

Final Project

• Create a complete CRISPR-Cas9 Experiment Plan for a target gene.
• Include: biological goal, target region, 3–5 gRNAs, predicted off-target strategy, delivery plan, controls, and validation workflow.
• Example projects: knockout a pathway regulator, tag a protein with GFP (concept), correct a point mutation (concept), screen design for a cancer gene.