Tips for Planning your Cas9 Experiment

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The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, several practical aspects should be carefully considered in order to achieve the best results from this system. Such considerations include: which promoter to use, whether to opt for wild-type of double nickase, how to achieve multiplexing and, perhaps most importantly, which vector to use.

For example, the chosen promoter may influence the range of target sites available (1). Using the U6 or T7 promoters requires a G or GG, respectively, at the 5′ end. Generating gRNAs with mismatches to the first two bases, or simply adding two guanines to the 5′ end, can reduce such restrictions.

Undoubtedly, the most important decision is to decide which vector to use. A variety of vectors have been validated for different cells and model organisms, and final application, from cutting or nicking to activating genes and screening libraries. Several groups have provided repositories of these plasmids, which are available through Addgene.


Examples of NEB products that can be used to support CRISPR workflows are shown below.
 
Product Name CRISPR/Cas9 Application NEB # Size
EnGen sgRNA Synthesis Kit Generation of microgram quantities of sgRNA E3322S 20 reactions
EnGen Mutation Detection Kit Determination of the targeting efficiency of genome editing protocols E3321S 25 reactions
EnGen Cas9 Nuclease NLS, S. pyogenes CRISPR/Cas9 Application Central component in the generation of CRISPR-based immunity - catalyzes site-specific cleavage of double-stranded DNA (same as product below) M0646T/M 400/2000 pmol
EnGen Spy Cas9 Nickase  Programmable site-specific DNA nicking for genome editing and in vitro applications  M0650S/T 70/400 pmol
EnGen Spy dCas9 (SNAP-tag)  Catalytically inactive Cas9 NLS with SNAP-tag for attachment of fluorophores, biotin, and a number of other conjugates  M0652S/T 70/400 pmol
Cas9 Nuclease, S. pyogenes Central component in the generation of CRISPR-based immunity - catalyzes site-specific cleavage of double-stranded DNA M0386S 50 rxns
Q5® Site-directed Mutagenesis Kit (with or without competent cells) Insertion of target sequence into the Cas9-sgRNA construct E0554S/E0552S 10 rxns
Q5® High-fidelity DNA Polymerases High-fidelity construct generation for use with CRISPR workflows Multiple Multiple
NEBuilder HiFi DNA Assembly Master Mix Single-tube, isothermal generation of the Cas9-sgRNA construct E2621S/L 10/50 rxns
NEBuilder HiFi DNA Assembly Cloning Kit Single-tube, isothermal generation of the Cas9-sgRNA construct E5520S 10 rxns
T7 Endonuclease I Determination of the targeting efficiency of genome editing protocols M0302S/L 250/1,250 units
HiScribe™ T7 High Yield RNA Synthesis Kit Generation of sgRNA E2040S 50 rxns
HiScribe™ T7 Quick High Yield RNA Synthesis Kit Generation of sgRNA E2050S 50 rxns
HiScribe™ SP6 RNA Synthesis Kit Generation of sgRNA E2070S 50 rxns
HiScribe™ T7 ARCA mRNA Kit (with tailing) Generation of capped Cas9 mRNA E2060S 20 rxns
HiScribe™ T7 ARCA mRNA Kit Generation of capped Cas9 mRNA E2065S 20 rxns
  1. Sander, J.D., and Joung, J.K. (2014) Nat Biotechnol. doi:10.1038/nbt.2842.