CRISPR Plasmids: Bacteria

The following CRISPR plasmids have been designed for use in bacteria.

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a 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. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

Base Edit

Catalytically dead dCas9 fused to a cytidine deaminase protein becomes a specific cytosine base editor that can alter DNA bases without inducing a DNA break. Cytosine base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the gRNA. Adenine base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite strand) mutations.

Nick

CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two gRNAs that target opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted off-target effects. Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed repair (HDR).

Prime Edit

Cas9 H840A nickase fused to a reverse transcriptase (RT) is capable of installing targeted insertions, deletions, and all possible base-to-base conversions using a prime editing guide RNA (pegRNA). The pegRNA directs the nickase to the target site by homology to a genomic DNA locus and encodes a primer binding site and the desired edits on an RT template.

Activate

Catalytically dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene of interest. 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 to your specific locus.

Interfere

Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression by interfering with transcription. Design your gRNA to target your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re using does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-repressor to your specific locus.

RNA Targeting

Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. In bacteria, once they have recognized and cleaved the target RNA sequence, they adopt an enzymatically active state and can bind and cleave additional RNAs regardless of homology to the crRNA. This activity provides a stark contrast to Cas9 and Cpf1, which require that each DNA target have high sequence identity to the spacer sequence and contain a PAM sequence just downstream of the sequence to be cleaved. This non-specific cleavage is thought to activate programmed cell death or dormancy for phage-infected bacterial cells so as to limit the spread of infection throughout the entire population.

Purify

A catalytically inactive Cas9 (dCas9) can be used to purify a region of genomic DNA and its associated proteins, RNA, and DNA. The enCHIP system uses an anti-FLAG antibody to immunoprecipitate FLAG-tagged Cas9. Design your gRNA sequence to direct dCas9 to a specific locus, avoiding known transcription factor and other protein binding sites.

	Plasmid	Gene/Insert	Promoter	PI	Publication
	3xFLAG-dCas9/p-bacteria	3xFLAG-dCas9	pLtetO-1	Fujii	Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun. 2013 Aug 11. pii: S0006-291X(13)01329-6. doi: 10.1016/j.bbrc.2013.08.013.

Empty gRNA Expression Vectors

Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids.

	gRNA Plasmid	Promoter	Cloning Enzyme(s)	Validated In	Resistance	Co-expressed Cas9	Depositing lab
Cas9 species = S. pyogenes (PAM = NGG)
	pCRISPR		BsaI	E. coli, S. pneumoniae	Kanamycin	none, need Cas9 plasmid	Marraffini
	pCas9		BsaI	E. coli, S. pneumoniae	Chloramphenicol	yes, cut	Marraffini
	pgRNA-bacteria	BBa_J23119	SpeI + HindIII		Ampicillin	none, need Cas9 plasmid	Qi