📋 Workflow
Full workflow, products add-to-cart directly1
sgRNA design and construction
1 dayDesign sgRNA with online tools, assess off-targets, clone into Cas9 expression vector
💡 Tip Choose high-specificity low off-target guides
⚠️ Caution Avoid targeting non-specific homology regions
🧪 Materials for this step (click to shop by spec/brand)
2
Delivery/transfection
1 dayTransfect cells (lipid/electroporation/nucleofection) or use RNP complex for higher efficiency
💡 Tip RNP reduces off-target effects
⚠️ Caution Passage cells to recover from stress
🧪 Materials for this step (click to shop by spec/brand)
3
Screen positive clones
1–2 weeksFluorescence sort or limiting dilution, pick and expand single clones
💡 Tip Single-cell cloning avoids mixed populations
⚠️ Caution Subclone verification for homozygous deletion
🧪 Materials for this step (click to shop by spec/brand)
4
Verify editing
1–2 weeksConfirm genotype by sequencing, verify knockout by western blot of protein
💡 Tip Cross-validate with multiple methods
⚠️ Caution Confirm both DNA and protein levels
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Off-target sgRNA causes non-specific editing
- ② Low delivery efficiency yields few positives
- ③ No single-cloning causes mixed editing
- ④ Unverified homozygous knockout confounds interpretation
- ⑤ Incomplete verification leaves protein expressed
❓ FAQ
+How to reduce off-target effects?
Choose specific guides, high-fidelity Cas9, or RNP delivery with short action.
+Incomplete knockout?
Use multiple guides, purify single clones, optimize with PAM/NHEJ.
+Can point-mutation knockin be done?
Yes, using an HDR donor template with homology arms.