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 | $400 | Add to Cart | |
| Kit | 1000000062 | Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack | 1 | $430 | Add to Cart | |
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
Figure from Sakuma et al., Sci Rep . 2014 Jun 23;4:5400.
Overview of Multiplex CRISPR dCas9/FokI-dCas9 plasmids
Figure adapted from Nakagawa et al., BMC Biotechnol. 2015 May 22;15(1):33.
Kit Documentation
Multiplex CRISPR-Cas9 Assembly System Kit.pdf 262.7Â KBProtocol
Download a PDF Protocol for Multiplex CRISPR construction as described in the above article.
Multiplex CRISPR Kit assembly protocol 365.9Â KBResources
Download a ZIP file containing Kit #1000000055 Multiplex CRISPR Kit plasmid sequences in GenBank format
Multiplex CRISPR Kit plasmids - GenBank files 126.1Â KBDownload a ZIP file containing Kit #1000000062 Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack plasmid sequences in GenBank format
Multiplex CRISPR dCas9/FokI-dCas9 Accessory Pack plasmid sequence GenBank files 88.9Â 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 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 |
Kit Plate Map - #1000000055
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 |