Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease.
Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S
Nat Biotechnol. 2013 Aug 8;31(8):691-3. doi: 10.1038/nbt.2655.
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PubMed
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Article
A CRISPR/Cas system designed and optimized for work in the model plant Nicotiana benthamiana .
Please refer to the Supplementary methods section of the manuscript for the protocol on assembling customised sgRNA expressing plasmids. Questions regarding the protocol can be addressed to [email protected] or [email protected].
Plasmids #49770, #49771 and #49772 are described in:
Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V. Plant Methods. 2013 Oct 11;9(1):39. PMID 24112467.
| ID | Plasmid |
|---|---|
| 48075 | pICH86966: A binary vector for plant gene expression (S. Marillonnet, described in Weber et al. (PLoS One 6:e16765, 2011). |
| 49770 | pICH41308::hCas9: Level 0 hCas9 module. |
| 49771 | pICH47742::2x35S-5'UTR-hCas9(STOP)-NOST: Level 1 hCas9 module. |
| 49772 | pAGM4723::AtU6p::sgRNA2-2x35S-5'UTR::Cas9::NOST-AtU6p::sgRNA1 : Level 2 construct. |
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 46965 | pK7WGF2::hCas9 | Expesses the human codon usage Cas9 nuclease (Mali et al. Science 339, 823-826, 2013) with an N-terminal GFP tag from the 35S promoter in the plant tissue |
| 46966 | pICH86966::AtU6p::sgRNA_PDS | Expresses an sgRNA targeting the PDS gene in Nicotiana benthamiana from the Arabidopsis U6 promoter |
| 46968 | pICSL01009::AtU6p | Encodes the Arabidopsis U6 promoter in a level 0 vector (Weber et al. PLoS One 6:e16765, 2011). It is used to place an sgRNA uder the Arabidopsis U6 promoter. |