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.

In nature, S. pyogenes Cas9 is programmed with two separate RNAs, the crRNA and tracrRNA. 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 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.