With so many options - how do you know which Cas9 format is best for your experiment?

The CRISPR-Cas9 system requires guide RNA and nuclease components to create a double-strand cut, and subsequent edit, to genomic DNA. Cas9 nuclease is available in multiple formats to allow for experimental flexibility and maximal success for particular cell types and applications. Since optimal activity of the RNA-guided Cas9 endonuclease is critical for successful gene editing, below is a list of considerations that can help direct you to the type of Cas9 nuclease best suited for your editing needs:

DNA-free Cas9: mRNA or protein

DNA-free Cas9 is most commonly used with synthetic guide RNA and chosen by researchers that would like to avoid unwanted vector DNA integration into their genomic DNA. Cas9 mRNA or protein is transiently available for editing events which means that these nuclease sources limit the off-targeting potential compared to constitutively expressed Cas9. For that reason, CRISPR-Cas9 utilizing mRNA or protein are ideal for applications such as knock in of a fluorescent reporter using HDR or knockout cell line generation. Both are amenable to lipid-based transfection reagents or electroporation to support a wide variety of cell types. 

Cas9 protein

The main advantage of delivering Edit-R Cas9 protein with synthetic guide RNA is that the translated protein is immediately available for complexing with the guide RNA and consequent editing in the cell. This is often a factor in applications where the cells lack robust translational machinery such as stem cells or embryonic model systems.

Cas9 mRNA

Purified, translation-ready Edit-R Cas9 nuclease mRNA, in combination with synthetic guide RNA is an economical option for researchers who want to perform DNA-free editing for generation of knockout or knock in clonal cell lines, or arrayed screening. Additionally, we offer a fluorescent version of Edit-R Cas9 mRNA to enrich for cells that are actively expressing Cas9 while still using a DNA-free system. 

Vector-based Cas9: plasmid or lentiviral particles

Vector-based Cas9 expression systems are useful for researchers who would like to enrich for Cas9-expressing cells or wish to establish a stable cell line. The Edit-R selection of vector-based Cas9 nuclease comes in two general formats:

Vector-based Cas9: promoter options

The activity of a particular promoter can result in variable Cas9 expression levels and potentially low editing efficiency. Vector-based Edit-R Cas9 Nuclease products are offered with a choice of six well-characterized constitutive cellular promoters so you can choose an optimal promoter for your cells. The Edit-R Lentiviral Cas9 Nuclease is also available with an inducible promoter to support creation of stable cell lines with minimal background expression, or for temporal control over Cas9 expression for wide-ranging experimental applications.


Additional Resources

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