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CRISPR header icon

Multiplex CRISPR/Cas9 Assembly Systems
(Kit # 1000000055, 1000000062 )

Depositing Lab: Takashi Yamamoto

These kits can be used to construct all-in-one CRISPR/Cas9 vectors expressing multiple gRNAs.

Kit #1000000055 is an 18 plasmid CRISPR kit used to express multiple gRNAs with either Cas9 nuclease or Cas9 D10A nickase.

This kit consists of one 96-well plate, and will be shipped as bacterial glycerol stocks on dry ice. Samples should be frozen at -80°C immediately upon arrival.

Kit #1000000062 is a separate accessory pack that contains plasmids for dCas9 and FokI-dCas9 expression. Please note that Kit #1000000055 is required to use these plasmids.

This kit consists of 12 individually packaged plasmids, which are shipped as bacterial stabs at room temperature.

Ordering

Item Catalog # Description Quantity Price (USD)
Kit 1000000055 Multiplex CRISPR/Cas9 Assembly System Kit 1 $330 Add to Cart
Kit 1000000062 Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack 1 $375 Add to Cart
This material is available to academics and nonprofits only.

Description

The Multiplex CRISPR/Cas9 Assembly System Kits enable construction of all-in-one CRISPR/Cas9 vectors expressing multiple gRNAs (2-7) and a Cas9 nuclease, nickase, dCas9, or dCas9-FokI fusion. Multiple gRNA cassettes are assembled using BsaI-mediated Golden Gate cloning method.

Publication List for Kits:

  • Kit #1000000055:
    Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. Sci Rep. 2014 Jun 23;4:5400. doi: 10.1038/srep05400. PubMed PMID 24954249.

  • Kit #1000000062:
    Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. Nakagawa Y, Sakuma T, Sakamoto T, Ohmuraya M, Nakagata N, Yamamoto T. BMC Biotechnol. 2015 May 22;15(1):33. doi:10.1186/s12896-015-0144-x. PubMed PMID 25997509.

Kit #1000000055: Multiplex CRISPR/Cas9 Assembly System Kit

This kit contains 18 plasmids: pX330A-1x[2-7], pX330A_D10A-1x[2-7], and pX330S-[2-7].

Kit #1000000062: Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack

The kit contains 12 plasmids: pX330A_dCas9-1x[2-7] and pX330A_FokI-1x[2-7].

Please note that Kit #1000000055 is required to use these plasmids.

Overview of Multiplex CRISPR/Cas9 assembly

Illustration of CRISPR all-in-one vector assembly

Figure from Sakuma et al., Sci Rep . 2014 Jun 23;4:5400.

Overview of Multiplex CRISPR dCas9/FokI-dCas9 plasmids

Figure dCas9-dCas9FokI assembly.png

Figure adapted from Nakagawa et al., BMC Biotechnol. 2015 May 22;15(1):33.

Kit Documentation

Icon Multiplex CRISPR-Cas9 Assembly System Kit.pdf (262.7 KB)

Protocol

Download a PDF Protocol for Multiplex CRISPR construction as described in the above article.

Icon Multiplex CRISPR Kit assembly protocol (365.9 KB)

Resources

Download a ZIP file containing Kit #1000000055 Multiplex CRISPR Kit plasmid sequences in GenBank format

Icon Multiplex CRISPR Kit plasmids - GenBank files (126.1 KB)

Download a ZIP file containing Kit #1000000062 Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack plasmid sequences in GenBank format

Icon Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack plasmid sequence GenBank files (88.9 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 CRISPR kit used for constructing multiplex CRISPR/Cas9 vectors was a gift from Takashi Yamamoto (Addgene kit # 1000000055) or (Addgene kit # 1000000062)"

For your Reference section:
Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. Sci Rep. 2014 Jun 23;4:5400. doi: 10.1038/srep05400. PubMed PMID 24954249.

Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. Nakagawa Y, Sakuma T, Sakamoto T, Ohmuraya M, Nakagata N, Yamamoto T. BMC Biotechnol. 2015 May 22;15(1):33. doi:10.1186/s12896-015-0144-x. PubMed PMID 25997509.

Multiplex CRISPR/Cas9 Assembly System Kit - #1000000055

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

Ampicillin
Spectinomycin

Inventory

Well Plasmid Resistance
A / 1 pX330A-1x2
Ampicillin
A / 2 pX330A-1x3
Ampicillin
A / 3 pX330A-1x4
Ampicillin
A / 4 pX330A-1x5
Ampicillin
A / 5 pX330A-1x6
Ampicillin
A / 6 pX330A-1x7
Ampicillin
A / 7 pX330A_D10A-1x2
Ampicillin
A / 8 pX330A_D10A-1x3
Ampicillin
A / 9 pX330A_D10A-1x4
Ampicillin
A / 10 pX330A_D10A-1x5
Ampicillin
A / 11 pX330A_D10A-1x6
Ampicillin
A / 12 pX330A_D10A-1x7
Ampicillin
B / 1 pX330S-2
Spectinomycin
B / 2 pX330S-3
Spectinomycin
B / 3 pX330S-4
Spectinomycin
B / 4 pX330S-5
Spectinomycin
B / 5 pX330S-6
Spectinomycin
B / 6 pX330S-7
Spectinomycin
Data calculated @ 2019-10-15

Kit Plate Map - #1000000055

Plate #1

  1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H

Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack - #1000000062

Kit #1000000062 consists of the following plasmids:

ID Name
63596 pX330A_dCas9-1x2
63597 pX330A_dCas9-1x3
63598 pX330A_dCas9-1x4
63599 pX330A_dCas9-1x5
63600 pX330A_dCas9-1x6
63601 pX330A_dCas9-1x7
63602 pX330A_FokI-1x2
63603 pX330A_FokI-1x3
63604 pX330A_FokI-1x4
63605 pX330A_FokI-1x5
63606 pX330A_FokI-1x6
63607 pX330A_FokI-1x7
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