CRISPR/Cas Plasmids for Use in Bacteria

Addgene is working with the leading scientists in the field to assemble the reagents and information you need to use the CRISPR/Cas9 technology in your own lab. The following Cas9 and gRNA expression plasmids have been designed for use in bacteria.

Cut

Fully functional Cas9 enzymes designed to introduce a double-strand break (DSBs) at a specific location based on a co-expressed gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ); a mechanism which frequently causes insertions or deletions (InDels) in the DNA, possibly resulting in frameshifts. If a repair template with high homology to the DNA surrounding the DSB is introduced along with the Cas9 and gRNA plasmids, the cell may instead repair the break using homology-directed repair (HDR). HDR is much less error-prone than NHEJ and can be used to faithfully introduce specific genomic changes.

Interfere

A catalytically inactive Cas9 (dCas9) or dCas9-repressor peptide fusion can be used to knock-down gene expression by interfering with transcription of the gene. Design your gRNA sequence to direct the dCas9 repressor to a specific genomic sequence. Potential target locations can include promoter regions, regulatory regions, and early coding regions. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9 repressor.

Activate

A catalytically inactive Cas9 (dCas9) fused to a transcription activator peptide can increase transcription of a gene. Design your gRNA sequence to direct the dCas9-activator to a specific genomic sequence. Potential target locations can include promoter regions and regulatory regions. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator.

Purify

A catalytically inactive Cas9 (dCas9) fused to an epitope tag(s) can be used to purify genomic DNA bound by the gRNA. Design your gRNA sequence to direct the dCas9 to a specific genomic sequence. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9.

Empty gRNA Expression Vectors

Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using the CRISPR/Cas system, you will need to express both a Cas9 protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas9 act as an all-in-one vector, but their function (cut, nick, activate, interfere...) is limited to that of the Cas9 present on the plasmid. gRNA plasmids that do not co-express Cas9 require a separate plasmid that does so; however, these independent gRNA plasmids can be paired with a wide variety of Cas9 plasmids and therefore are not limited to a single Cas9 function.