zCas9i cloning kit: MoClo-compatible CRISPR/Cas9 cloning kit with intronized Cas9
(Kit #
1000000171
)
Depositing Lab: Sylvestre Marillonnet and Johannes Stuttmann
This collection provides a series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs for use in plants. The constructs are made with zCas9i, a high efficiency intron-optimized SpCas9-coding gene.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publications
High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns Grützner R, Martin P, Horn C, Mortensen S, Cram EJ, Lee-Parsons CWT, Stuttmann J, Marillonnet S. Plant Communications 2021 Volume 2, Issue 2 doi: 10.1016/j.xplc.2020.100135. Publication.
Highly efficient multiplex editing: One-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants Stuttmann J, Barthel K, Martin P, Ordon J, Erickson JL, Herr R, Ferik F, Kretschmer C, Berner T, Keilwagen J, Marillonnet S, Bonas U. Plant J. 2021 Feb 12. doi: 10.1111/tpj.15197. Epub ahead of print. PubMed PMID: 33577114.
Description
This collection provides a series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs for use in plants. The constructs are made with zCas9i, a high efficiency intron-optimized SpCas9-coding gene.
A first set of vectors and parts is provided for assembly of CRISPR/Cas9 constructs using the modular cloning system MoClo. Some of the vectors provided contain all CRISPR components except a guide RNA, which can be cloned by ligation of 2 annealed oligonucleotide primers. Individual CRISPR vector components are also provided to allow users to prepare constructs containing their promoter of choice and desired number of guide RNAs. Preparation of these constructs requires some components of the MoClo Toolkit (Kit # 1000000044).
A second set of “pDGEvectors” is intended for rapid, PCR-free assembly of Cas9 constructs containing up to 32 guide RNA transcriptional units. The system provides a choice of three different RNA polymerase III promoters for guide RNA expression. After construction, guide RNA transcriptional units are assembled in recipient plasmids readily containing the zCas9i gene and additional positive and negative selection markers.
Construction of CRISPR constructs with single (A) or multiple guide RNAs (B).
Kit Documentation
Protocols for use are provided in the publications associated with the collection cited below, and the supplementary material of these publications. This includes a detailed description on how to generate CRISPR/Cas vectors containing zCas9i by MoClo (1st set of plasmids, Grützner et al: Figures S14-17) and a laboratory manual for using pDGE plasmids (2nd set of plasmids, Barthel et al: Appendix 1). See the PDF below for an overview of the plasmids included in this kit.
Kit Overview.pdf 333.5 KBHow to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The zCas9i cloning kit: MoClo-compatible CRISPR/Cas9 cloning kit with intronized Cas9 was a gift from Sylvestre Marillonnet and Johannes Stuttmann (Addgene kit #1000000171).”
For your Reference section:
High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns Grützner R, Martin P, Horn C, Mortensen S, Cram EJ, Lee-Parsons CWT, Stuttmann J, Marillonnet S. Plant Communications 2021 Volume 2, Issue 2 doi: 10.1016/j.xplc.2020.100135. Publication.
Highly efficient multiplex editing: One-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants Stuttmann J, Barthel K, Martin P, Ordon J, Erickson JL, Herr R, Ferik F, Kretschmer C, Berner T, Keilwagen J, Marillonnet S, Bonas U. Plant J. 2021 Feb 12. doi: 10.1111/tpj.15197. Epub ahead of print. PubMed PMID: 33577114.
zCas9i cloning kit: MoClo-compatible CRISPR/Cas9 cloning kit with intronized Cas9 - #1000000171
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Spectinomycin | |
| Ampicillin | |
| Chloramphenicol and Spectinomycin | |
| Kanamycin | |
| Chloramphenicol and Ampicillin | |
| Chloramphenicol and Kanamycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pAGM41991 | Spectinomycin |
| A / 2 | pAGM38869 | Spectinomycin |
| A / 3 | pAGM47523 | Spectinomycin |
| A / 4 | pAGM9037 | Spectinomycin |
| A / 5 | pAGM35171 | Ampicillin |
| A / 6 | pICH67131 | Ampicillin |
| A / 7 | pAGM51323 | Ampicillin |
| A / 8 | pDGE347 | Chloramphenicol and Spectinomycin |
| A / 9 | pDGE651 | Chloramphenicol and Spectinomycin |
| A / 10 | pDGE652 | Chloramphenicol and Spectinomycin |
| A / 11 | pDGE666 | Chloramphenicol and Spectinomycin |
| A / 12 | pDGE667 | Chloramphenicol and Spectinomycin |
| B / 1 | pDGE668 | Chloramphenicol and Spectinomycin |
| B / 2 | pDGE463 | Chloramphenicol and Spectinomycin |
| B / 3 | pDGE669 | Chloramphenicol and Spectinomycin |
| B / 4 | pDGE670 | Chloramphenicol and Spectinomycin |
| B / 5 | pDGE671 | Chloramphenicol and Spectinomycin |
| B / 6 | pDGE672 | Chloramphenicol and Spectinomycin |
| B / 7 | pDGE673 | Chloramphenicol and Spectinomycin |
| B / 8 | pAGM55261 | Kanamycin |
| B / 9 | pAGM55273 | Kanamycin |
| B / 10 | pAGM55285 | Kanamycin |
| B / 11 | pAGM55297 | Kanamycin |
| B / 12 | pAGM65879 | Kanamycin |
| C / 1 | pAGM65881 | Kanamycin |
| C / 2 | pAGM65893 | Kanamycin |
| C / 3 | pAGM65905 | Kanamycin |
| C / 4 | pAGM37443 | Kanamycin |
| C / 5 | pAGM51559 | Kanamycin |
| C / 6 | pAGM55361 | Kanamycin |
| C / 7 | pDGE508 | Kanamycin |
| C / 8 | pDGE509 | Kanamycin |
| C / 9 | pDGE510 | Kanamycin |
| C / 10 | pDGE331 | Chloramphenicol and Ampicillin |
| C / 11 | pDGE332 | Chloramphenicol and Ampicillin |
| C / 12 | pDGE333 | Chloramphenicol and Ampicillin |
| D / 1 | pDGE334 | Chloramphenicol and Ampicillin |
| D / 2 | pDGE335 | Chloramphenicol and Ampicillin |
| D / 3 | pDGE336 | Chloramphenicol and Ampicillin |
| D / 4 | pDGE337 | Chloramphenicol and Ampicillin |
| D / 5 | pDGE495 | Chloramphenicol and Ampicillin |
| D / 6 | pDGE496 | Chloramphenicol and Ampicillin |
| D / 7 | pDGE497 | Chloramphenicol and Ampicillin |
| D / 8 | pDGE341 | Chloramphenicol and Ampicillin |
| D / 9 | pDGE342 | Chloramphenicol and Ampicillin |
| D / 10 | pDGE654 | Chloramphenicol and Ampicillin |
| D / 11 | pDGE655 | Chloramphenicol and Ampicillin |
| D / 12 | pDGE656 | Chloramphenicol and Ampicillin |
| E / 1 | pDGE657 | Chloramphenicol and Ampicillin |
| E / 2 | pDGE658 | Chloramphenicol and Ampicillin |
| E / 3 | pDGE659 | Chloramphenicol and Ampicillin |
| E / 4 | pDGE660 | Chloramphenicol and Ampicillin |
| E / 5 | pDGE661 | Chloramphenicol and Ampicillin |
| E / 6 | pDGE662 | Chloramphenicol and Ampicillin |
| E / 7 | pDGE663 | Chloramphenicol and Ampicillin |
| E / 8 | pDGE664 | Chloramphenicol and Ampicillin |
| E / 9 | pDGE665 | Chloramphenicol and Ampicillin |
| E / 10 | pDGE473 | Chloramphenicol and Ampicillin |
| E / 11 | pDGE474 | Chloramphenicol and Ampicillin |
| E / 12 | pDGE475 | Chloramphenicol and Ampicillin |
| F / 1 | pDGE476 | Chloramphenicol and Ampicillin |
| F / 2 | pDGE477 | Chloramphenicol and Ampicillin |
| F / 3 | pDGE478 | Chloramphenicol and Ampicillin |
| F / 4 | pDGE479 | Chloramphenicol and Ampicillin |
| F / 5 | pDGE498 | Chloramphenicol and Ampicillin |
| F / 6 | pDGE499 | Chloramphenicol and Ampicillin |
| F / 7 | pDGE500 | Chloramphenicol and Ampicillin |
| F / 8 | pDGE501 | Chloramphenicol and Ampicillin |
| F / 9 | pDGE502 | Chloramphenicol and Ampicillin |
| F / 10 | pDGE503 | Chloramphenicol and Kanamycin |
| F / 11 | pDGE504 | Chloramphenicol and Kanamycin |
| F / 12 | pDGE505 | Chloramphenicol and Kanamycin |
| G / 1 | pDGE506 | Chloramphenicol and Kanamycin |
Kit Plate Map - #1000000171
Accessory Plasmids
Five additional accessory plasmids are also available. These plasmids are not included in the kit, but can be ordered separately:
- pDGE687: Vector for plant genome editing containing p35S:GFP-Cas9 expression cassette, nptII selection marker, and SlFAST2 and Ca-Bs3 counterselction markers, but not sgRNAs.
- pDGE792: Vector for plant genome editing containing pRPS5a:GFP-Cas9 expression cassette, Bar (BASTA/PPT) selection marker, and FCY-UPP and Ca-Bs3 counterselction markers, but not sgRNAs.
- pJOG1368: Level 0 module (CDS1) for further assemblies based on the Modular Cloning system. FCY-UPP gene conferring sensitivity to 5-Fluorocytosine.
- pJOG1334: Level 0 module (Pro+5U) for further assemblies based on the Modular Cloning system. Promoter and 5'UTR of a Solanum lycopersicum Oleosin gene, arbitrarily called OLE2 (Sollyc03g112440).
- pJOG1336: Level 0 module (3U+Ter) for further assemblies based on the Modular Cloning system. 3'UTR and flanking sequence of a Solanum lycopersicum Oleosin gene, arbitrarily called OLE2 (Sollyc03g112440).