EnGen® sgRNA Synthesis Kit, S. pyogenes
- Rapid generation of microgram quantities of sgRNA in under an 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 - now included in the NEBioCalculator® Tool
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.
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.
1. Jinek, M. et al. (2012) Science 816–821. PubMed ID: 22745249.
Figure 1: General workflow for the EnGen sgRNA Synthesis Kit, S. pyogenes.
Figure 2: EnGen sgRNA Synthesis Kit Overview
Figure 3: Examples of sgRNAs synthesized using the EnGen sgRNA Synthesis Kit, S. pyogenes and multiple different target-specific oligos including the EnGen sgRNA Control Oligo, S. pyogenes.
The following reagents are supplied with this product:
Store at (°C) Concentration DNase I (RNase-free) -20 2 units/μl EnGen® sgRNA Enzyme Mix -20 EnGen® 2X sgRNA Reaction Mix, S. pyogenes -20 EnGen® sgRNA Control Oligo, S. pyogenes -20
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