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CRISPR-Cas9 Variants
1. Streptococcus pyogenes Cas9 (SpCas9)
⭐ The gold standard and most widely used Cas9
- Size: ~160 kDa
- PAM: NGG (highly abundant in genomes)
- Why it’s popular:
- Very high editing efficiency
- Extensive validation and optimization across cell lines and organisms
- Broad availability of plasmids, proteins, antibodies, and protocols
- Common formats: plasmid, mRNA, RNP
- Typical use: standard knockout/knock-in experiments
2. High-Fidelity SpCas9 Variants
Used when off-target reduction is critical.
- SpCas9-HF1
- eSpCas9 (1.1)
- HypaCas9
- HiFi Cas9 protein
- PAM: NGG
- Use case: therapeutic research, sensitive functional genomics
3. Staphylococcus aureus Cas9 (SaCas9)
- Size: ~105 kDa (smaller than SpCas9)
- PAM: NNGRRT
- Why it’s used:
- Fits more easily into AAV vectors
- Trade-off: more restrictive PAM than SpCas9
- Common in: in vivo and gene-therapy applications
4. Cas9 Nickase (Cas9n)
- Mutations: D10A or H840A
- Function: cuts only one DNA strand
- Use: paired-gRNA strategies to reduce off-target effects
5. Dead Cas9 (dCas9)
- Catalytically inactive
- Applications:
- CRISPRi (gene repression)
- CRISPRa (gene activation)
- Epigenome editing
- Live-cell imaging
Quick Summary Table
| Cas9 Variant | PAM | Key Advantage | Common Use |
|---|---|---|---|
| SpCas9 | NGG | Highest versatility | General genome editing |
| SpCas9-HF | NGG | Low off-target | Precision editing |
| SaCas9 | NNGRRT | Small size | AAV delivery |
| Cas9n | NGG | Fewer off-targets | Paired cutting |
| dCas9 | NGG | No cutting | Gene regulation |
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