Product Class: Kit

EnGen® sgRNA Synthesis Kit, S. pyogenes

Note: Kit and protocol have been updated. Please visit Protocols & Manuals tab for details.

Product Introduction

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.


Catalog # Size Concentration
E3322S 20.0 reactions

Product Information

Description







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

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. 

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.

RNA was run under denaturing conditions on a 10% Novex TBE-Urea gel and post-stained with SYBR Gold.
Figure 4. Comparison of synthetic RNA with 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)
This product is related to the following categories:
sgRNA Synthesis Reagents and Kits Products,
Genome Editing Products,
This product can be used in the following applications:
Genome Editing Applications

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E3322S     -20    
      EnGen® sgRNA Enzyme Mix M3322AVIAL -20 1 x 0.04 ml Not Applicable
      EnGen® 2X sgRNA Reaction Mix, S. pyogenes N3320AVIAL -20 1 x 0.2 ml Not Applicable
      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

Protocols, Manuals & Usage

Protocols

  1. EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (NEB #E3322)
  2. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. The sgRNA is not running as a single band on the gel – why is this happening?
  2. Is it necessary to add a “G” into the oligo following the T7 promoter sequence?
  3. Is it necessary to DNase treat my sgRNA synthesis reaction?
  4. Why is my sgRNA not exhibiting 100% in vitro Cas9 cleavage activity?
  5. Is it necessary to phosphatase treat my sgRNAs?
  6. The sgRNA is not running as a single band on the gel – why is this happening?
  7. Will the sgRNAs synthesized from this kit (NEB #E3322S) be recognized by homologs of Cas9 from different organisms?
  8. Do I include the PAM(NGG) in my sgRNA sequence?
  9. What is the sequence of the control oligo provided with the EnGen sgRNA Synthesis Kit, S. pyogenes?
  10. Is the Monarch Spin RNA Cleanup Kit (NEB #T2040) compatible with the EnGen sgRNA Synthesis Kit, S. pyogenes?
  11. Is it necessary to add the provided DTT to the sgRNA synthesis reaction?

Quality, Safety & Legal

Quality Assurance Statement

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]

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 DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

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