Skip to main content
This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies.

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more

Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at [email protected] Learn more

plasmid kit icon

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.

Add to Cart
$375 USD + shipping

Available to academics and nonprofits only.

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 KB

How 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
Data cached @ 2021-10-22

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).
Content blocked, you may need to disable your ad-blocker.