Home › Genetics / Genomics › CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation
Genetics / Genomics JoVE (Open Access) Citable · DOI

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

DOI: 10.3791/59512-v
What you'll learn
  • ✓ Prepare mammalian cells for efficient CRISPR/Cas9 RNP delivery
  • ✓ Execute tube electroporation with Cas9 ribonucleoprotein complexes
  • ✓ Analyze and quantify gene editing outcomes in treated cells
Protocol

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Difficulty
intermediate
Total time
~4–6 hours (cell preparation through analysis)
Model organism
Mammalian cell culture (HEK293 or equivalent)
Biosafety
BSL-1

Steps

1
Prepare cells for electroporation

Culture and harvest mammalian cells at appropriate confluence and viability. Resuspend cells in electroporation buffer to achieve optimal cell density for tube electroporation.

▶ 01:08
2
Deliver Cas9 RNP and ssODN via electroporation

Combine Cas9 ribonucleoprotein complex and single-stranded oligodeoxynucleotide templates with prepared cells in a tube cuvette. Apply optimized electrical pulses to introduce the RNP complex into cells.

▶ 02:51
3
Quantify gene editing by molecular analysis

Extract genomic DNA and perform sequencing or PCR-based analysis to detect indels and precise insertions. Measure editing efficiency and characterize repair outcomes.

▶ 04:24
4
Evaluate tube electroporation results

Review quantitative editing data and cell viability metrics from treated populations. Assess protocol performance and refine parameters if needed.

▶ 05:48
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