Catalytically dead Streptococcus pyogenes Cas9 (dCas9)

Catalytically inactive Cas9 that binds DNA at guide-specified sites without cleaving it, produced by the D10A and H840A substitutions in the RuvC and HNH nuclease domains of Streptococcus pyogenes Cas9. Retains 20-nucleotide guide targeting and the NGG PAM requirement, and serves as the programmable DNA-binding scaffold for CRISPRi, CRISPRa, and epigenome editing fusions.

Length: 4107 bp (1369 aa)

PAM: NGG

Origin: Engineered from Streptococcus pyogenes Cas9 (D10A/H840A)

Characteristics

Binds double-stranded DNA at sites matched by a 20-nucleotide guide sequence next to an NGG PAM but cleaves neither strand. D10A inactivates the RuvC-like domain, which cuts the noncomplementary strand, and H840A inactivates the HNH domain, which cuts the guide-complementary strand; either substitution on its own leaves a nickase that cuts one strand. Binding is reversible and leaves no mutation at the target site. Bound dCas9 blocks transcription by steric occlusion alone. In Escherichia coli, a single sgRNA against the nontemplate strand of a reporter open reading frame gives 10- to 300-fold repression and two sgRNAs together up to 1,000-fold, while the same approach in HEK293 cells gives only 46-63% repression without a fused repressor domain.

Applications: CRISPRi with a fused KRAB repressor reaches 90-99% knockdown of endogenous human genes, and genome-scale CRISPRi and CRISPRa libraries covering every annotated human gene support pooled screening for growth and toxin sensitivity. CRISPRa fusions to VP64, the tripartite VPR activator, or SunTag-recruited activators raise target expression 10- to 10,000-fold. Epigenome editing fusions deposit H3K27ac (dCas9-p300) or CpG methylation (dCas9-Dnmt3a-Dnmt3L), or remove methylation in vivo (dCas9-TET1).

Limitations: CRISPRi repression requires the guide to land between -50 and +300 bp of the transcription start site, and within an open reading frame only nontemplate-strand guides repress. Each sgRNA also directs dCas9 to tens to thousands of off-target sites in mouse embryonic stem cells, needing only a 5-nucleotide PAM-proximal seed match plus an NGG PAM; 70% lie within genes. The original CRISPRi dCas9 carries H841A, not H840A.

Sequence

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References

  1. Jinek et al. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science - Jinek 2012 SpCas9
  2. Mali et al. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol - Mali 2013 Cas9 Nickase
  3. Gilbert et al. (2014). Genome-scale CRISPR-mediated control of gene repression and activation. Cell - Gilbert 2014 CRISPR Screen
  4. Chavez et al. (2015). Highly efficient Cas9-mediated transcriptional programming. Nat Methods - Chavez 2015 VPR
  5. Tanenbaum et al. (2014). A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell - Tanenbaum 2014 SunTag
  6. Hilton et al. (2015). Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol - Hilton 2015 p300
  7. Stepper et al. (2017). Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase. Nucleic Acids Res - Stepper 2017 DNMT3A
  8. Morita et al. (2016). Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. Nat Biotechnol - Morita 2016 TET1

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