CRISPR Plasmids: Plants

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

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.

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.

	Plasmid	Promoter	Cloning Enzyme	Co-expressed Cas9	Cas9 System	Selection	PI
	pICSL01009::AtU6p	aU6	BsaI	none	S. pyogenes		Kamoun
	pUC119-gRNA	U6	PCR template	none	S. pyogenes		Sheen
	pRGEB31	rice snoRNA U3	BsaI	none	S. pyogenes		Yang
	pBUN6A11	OsU3	BsaI	yes, activate	S. pyogenes	Bar	Chen
	pBUN6I11	OsU3	BsaI	yes, interfere	S. pyogenes	Bar	Chen
	pBUN501	AtU6-26	BsaI	yes, nick	S. pyogenes	Bar	Chen
	pCBC-MT2T3	see paper	BsaI	none	S. pyogenes		Chen
	pCBC-MT3T4	see paper	BsaI	none	S. pyogenes		Chen
	pBUN421	TaU3	BsaI	yes, cut	S. pyogenes	Bar	Chen
	pU3-gRNA	OsU3	AarI	none	S. pyogenes		Gao
	pZmU3-gRNA	maize U3		none	S. pyogenes		Gao
	pU6-gRNA	wheat U6	BbsI	none	S. pyogenes		Gao
	pBlu/gRNA	Arabidopsis U6	BbsI	none	S. pyogenes		Stupar
	pBUE411	OsU3		yes, cut	S. pyogenes	Basta	Chen
	pHUE411	OsU3		yes, cut	S. pyogenes	Hyg	Chen
	pHAtC	U6	AarI	yes, cut	S. pyogenes	Hygro	Kim
	pBAtC	U6	AarI	yes, cut	S. pyogenes	Basta	Kim
	pHEE401	U6-26p	BsaI	yes, cut	S. pyogenes	Hygro	Chen
	pHEE401E	U6-26p	BsaI	yes, cut	S. pyogenes	Hygro	Chen
	pHDE-35S-Cas9-mCherry-UBQ	U6-26	PmeI	yes, cut	S. pyogenes	Hygro, mCherry	Zhao
	pCBC-DT1T1	see paper	BsaI	none	S. pyogenes		Chen
	pRGEB32	rice snoRNA U3	BsaI	yes, cut	S. pyogenes	Hygro	Yang
	pHSE401	AtU6-26		yes, cut	S. pyogenes	Hyg	Chen
	pBUN411	OsU3	BsaI	yes, cut	S. pyogenes	Bar	Chen
	pHSN6A01	AtU6-26	BsaI	yes, activate	S. pyogenes	Hyg	Chen
	pHSN6I01	AtU6-26	BsaI	yes, interfere	S. pyogenes	Hyg	Chen
	pHSN501	AtU6-26	BsaI	yes, nick	S. pyogenes	Hyg	Chen
	pCBC-DT3T4	see paper	BsaI	none	S. pyogenes		Chen
	pCBC-MT1T2	see paper	BsaI	none	S. pyogenes		Chen
	pRGE32	rice snoRNA U3	BsaI	yes, cut	S. pyogenes	Hygro	Yang
	pKSE401	AtU6-26		yes, cut	S. pyogenes	Kan	Chen
	pRGE31	rice snoRNA U3	BsaI	none	S. pyogenes		Yang
	pUC gRNA Shuttle	Mt U6.6	inFusion	none	S. pyogenes		Parrott
	pCBC-DT2T3	see paper	BsaI	none	S. pyogenes		Chen
	pHSN401	AtU6-26	BsaI	yes, cut	S. pyogenes	Hyg	Chen
	pYPQ141-ZmUbi-RZ-As	Maize ubiquitin 1		none	As Cpf1		Qi
	pKEE401			yes, cut	S. pyogenes	Neomycin	Chen