Genome editing (CRISPR/Cas9)

Cas9 Nuclease protein

The CRISPR/Cas genome editing system consists of a “guide” RNA (gRNA) and the Cas9 endonuclease. The gRNA is comprised of a synthetic RNA with a “scaffold” sequence to which Cas9 binds as well as a user-selected ∼20 nucleotide “targeting” sequence for the genomic target to be modified. The Cas9 protein and gRNA form a complex through interaction between the gRNA scaffold domain and positively-charged Cas9 grooves. Recombinant Cas9 Nuclease protein NLS which forms a complex with in vitro transcribed guide RNAs cleaves the target sequence, serving as an ideal tool to edit eukaryotic genomes.

Cas9 Nuclease protein NLS, in complex with guide RNA, offers benefits for CRISPR/Cas9 technology over plasmid or mRNA transfection:

• Off-target mutations rarely occur
  Prolonged expression of Cas9 increases off-target editing but RNP delivery is naturally transient

• Transfection/injection is easy
  Cas9-NLS bypasses transcription and translation by delivering the protein directly

• The efficiency of the guide RNA in vitro is measurable
  The RNP complex can be tested with a cleavage assay in vitro before it is used in cell culture or in vivo
  
  
• Assay is DNA-free
  No risk of DNA integration that can happen using plasmids plus no cloning required

Efficient mutagenesis of endogenous gene in zebrafish by microinjection of Cas9 Nuclease protein NLS.

Mutagenesis of a gene required for angiogenesis in the brain using Cas9 Nuclease protein NLS and a specific sgRNA. Maximal intensity projection of a confocal z-stack of the cranial vasculature of Tg(kdrl:GFP) zebrafish embryos at 4dpf in dorsal view (anterior to the left). (A) Uninjected control sibling and (B) embryos injected with Cas9 Nuclease protein NLS (300pg) and sgRNA (30pg) RNP complex. On average, 85% of the injected embryos display a total absence of hindbrain intracerebral blood vessels at 4dpf. This knock-out causes specific CNS vascular defects, providing a visual mean that the gene of interest in inactivated by the CRISPR/Cas9 system.

Protocols

• Transfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine® RNAiMAX
• CRISPR/Cas9 editing: mutation detection with mismatch cleavage assay
• Selection of guide RNA by ChIP

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