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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.

Plasmid Gene/Insert Selectable Marker PI Publication

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.

Plasmid Gene/Insert Promoter Selectable Marker PI Publication

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).

Plasmid Gene/Insert Promoter Selectable Marker PI Publication

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.

Plasmid Gene/Insert Promoter Selectable Marker PI Publication

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.

Plasmid Gene/Insert Promoter Selectable Marker PI Publication

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

Do you have suggestions for other plasmids that should be added to this list?

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