EnGen® sgRNA Synthesis Kit, S. pyogenes

Catalog #	Concentration	Size	List Price	Quantity	Your Price
E3322S		20 reactions	$683.00		$614.70
E3322V		10 reactions	$354.00		$318.60

Catalog #	Size	List Price	Your Price
E3322S	20 reactions	$683.00	$614.70
E3322V	10 reactions	$354.00	$318.60

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.

Arrow showing the 5 steps of the EnGen sgRNA Synthesis Kit workflow

Workflow for the EnGen sgRNA Synthesis Kit showing the original oligo and the single-tube reaction — A. The target-specific oligo contains the T7 promoter sequence, ~20 nucleotides of target-specific sequence and a 14 nucleotide overlap sequence complementary to the S. pyogenes Cas9 Scaffold Oligo supplied in the reaction mix. Target-specific oligos (or EnGen sgRNA Control Oligo, S. pyogenes) are mixed with the EnGen 2X sgRNA Reaction Mix (NTPs, dNTPs, S. pyogenes Cas9 Scaffold Oligo), 0.1 M DTT and the EnGen sgRNA Enzyme Mix (DNA and RNA polymerases) at room temperature. B. At 37°C the two oligos anneal at the 14 nucleotide overlap region of complementarity. C. The DNA polymerase extends both oligos from their 3´ ends creating a double-stranded DNA template. D. The RNA polymerase recognizes the double-stranded DNA of the T7 promoter and initiates transcription. The resulting sgRNA contains the target-specific/crRNA sequence as well as the tracrRNA. All steps occur in a single reaction during a 30 minute incubation at 37°C.

Gel showing sgRNAs synthesized using the EnGen sgRNA Synthesis Kit, S. pyogenes and multiple different target-specific oligos — RNA was run under denaturing conditions on a 10% Novex TBE-Urea gel and post-stained with SYBR Gold.

Graph showing sgRNA Synthesis comparison with synthetic in vitro transcribed RNA — 293T cells were electroporated using Lonza SF Cell Line 4D-Nucleofector™ Kit. 48 hours later, DNA was extracted using Epicentre QuickExtract™. Percent modification was determined using the EnGen Mutation Detection Kit (NEB #E3321S)

Reagents Supplied

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

E3322V -20

EnGen^® sgRNA Enzyme Mix	M3322AVIAL	-20	1 x 0.04 ml
EnGen^® 2X sgRNA Reaction Mix, S. pyogenes	N3320SVIAL	-20	1 x 0.1 ml
DNase I (RNase-free)	M0303AAVIAL	-20	1 x 0.04 ml	2,000 units/ml
EnGen^® sgRNA Control Oligo, S. pyogenes	S3321AVIAL	-20	1 x 0.02 ml	1 µM
Dithiothreitol (DTT)	B1222AVIAL	-20	1 x 0.5 ml	100 mM

E3322S -20

EnGen^® sgRNA Enzyme Mix	M3322AVIAL	-20	1 x 0.04 ml
EnGen^® 2X sgRNA Reaction Mix, S. pyogenes	N3320AVIAL	-20	1 x 0.2 ml
DNase I (RNase-free)	M0303AAVIAL	-20	1 x 0.04 ml	2,000 units/ml
EnGen^® sgRNA Control Oligo, S. pyogenes	S3321AVIAL	-20	1 x 0.02 ml	1 µM
Dithiothreitol (DTT)	B1222AVIAL	-20	1 x 0.5 ml	100 mM

Properties & Usage

Materials Required but not Supplied

Target-specific DNA oligo(s)
Thermocycler/37°C heat block/incubator
Nuclease-free water
Equipment and reagents for RNA quantitation
Spin columns for RNA cleanup (e.g. NEB #T2040)
RNase-free tubes, aerosol tips
Microcentrifuge

Optional Materials:
Alkaline Phosphatase, Calf Instestinal (CIP)
Cas9 Nuclease, S. pyogenes
EnGen Spy Cas9, NLS
Gels, running buffer, gel loading dye, RNA and DNA ladders, gel box

Companion Products

Protocols

DataCards

manualE3322

Web Tools

EnGen sgRNA Template Oligo Designer

FAQs

Citations

See more details on Bioz

Additional Citations

Evan A. Boyle, Johan O.L. Andreasson, Lauren M. Chircus, Samuel H. Sternberg, Michelle J. Wu, Chantal K. Guegler, Jennifer A. Doudna, and William J. Greenleaf (2017) High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding Proc Natl Acad Sci U S A 114, 5461-5466.

Publications

Gertsenstein, M., & Nutter, L. M. J. (2018). Engineering point mutant and epitope-tagged alleles in mice using Cas9 RNA-guided nuclease Curr Protoc Mouse Biol.PubMedID: 30040228

Quality Control Assay

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

E3322S_v2

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety Data Sheets

Legal And Disclaimer

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.