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broad-institute/CRISPR-Cas9 Knockout

Demo
Advanced Molecular Biology 18 steps ~5 days v2.1
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Gene knockout using CRISPR-Cas9 ribonucleoprotein delivery in mammalian cells.

This protocol covers CRISPR-Cas9-mediated gene knockout in mammalian cell lines using ribonucleoprotein (RNP) delivery. RNP electroporation offers advantages over plasmid-based approaches including reduced off-target effects, no risk of genomic integration, and faster editing kinetics.

Overview

The workflow spans guide RNA design and validation, RNP complex assembly, electroporation into target cells, clonal expansion, and genotyping to confirm knockout. Typical editing efficiencies of 70–90% are achievable at most loci, with successful biallelic knockout rates of 30–60%.

Key considerations

  • Guide design: Use 2–3 guides per target gene; select guides with high on-target scores and minimal off-target predictions
  • Cell type optimization: Electroporation parameters vary by cell line; optimize pulse conditions before scaling
  • Timing: Day 1: RNP assembly + electroporation; Days 2–4: recovery; Day 5+: genotyping and clonal isolation
  • Validation: Confirm knockout at both DNA (T7E1 assay, Sanger sequencing) and protein (Western blot) levels

Materials

Alt-R S.p. Cas9 Nuclease V3 (IDT, cat. 1081058)
Alt-R CRISPR-Cas9 crRNA (custom design, IDT)
Alt-R CRISPR-Cas9 tracrRNA (IDT, cat. 1072532)
Nuclease-free Duplex Buffer (IDT)
Neon Transfection System (Invitrogen)
T7 Endonuclease I (NEB, cat. M0302)
Genomic DNA extraction kit

Tags

CRISPR Cas9 gene editing knockout RNP