In nature, S. pyogenes Cas9 is programmed with two separate RNAs, the crRNA and tracrRNA.
- Programmed with two RNAs and contains approximately 20 nucleotides
- Adapted for use in the lab
- Create double-stranded DNA for transcription
- Rapid generation of microgram quantities of sgRNA
- Complete workflow of less than one hour
- Simple protocol requires only a user-supplied ~55 nucleotide ssDNA target-specific oligonucleotide
- For help with oligo design, try EnGen sgRNA Template Oligo Designer
The crRNA, or CRISPR RNA sequence contains approximately 20 nucleotides of homology complementary to the strand of DNA opposite and upstream of a PAM (Protospacer Adjacent Motif) (NGG) sequence
The EnGen sgRNA Synthesis Kit, S. pyogenes combines an S. pyogenes Cas9-specific Scaffold Oligo (included in the EnGen 2X sgRNA Reaction Mix) that is partially complementary to the target-specific oligos designed by the user. The two oligos anneal at the overlapping region and are filled in by the DNA polymerase, creating a double-stranded DNA (dsDNA) template for transcription. Synthesis of the dsDNA template and transcription of RNA occur in a single reaction, resulting in the generation of a functional sgRNA.
The tracrRNA, or transactivating crRNA, contains partial complementary sequence to the crRNA as well as the sequence and secondary structure that is recognized by Cas9. These sequences have been adapted for use in the lab by combining the tracrRNA and crRNA into one long single guide RNA (sgRNA) (1) species capable of complexing with Cas9 to recognize and cleave the target DNA.
The EnGen sgRNA synthesis kit, S. pyogenes provides a simple and quick method for transcribing high yields of sgRNA in a single 30-minute reaction, using the supplied reagents and target-specific DNA oligos designed by the user.