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|
- Sander, J.D., and Joung, J.K. (2014) Nat Biotechnol. doi:10.1038/nbt.2842.