CRIMPkit
(Kit #
1000000225
)
Depositing Lab: Robert Bryson-Richardson
The CRISPR/Cas9 Insertional Mutagenesis Protocol (CRIMP) and toolkit (CRIMPkit) is a universal and highly efficient targeted insertional mutagenesis system designed for use in zebrafish.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
CRIMP: A CRISPR/Cas9 Insertional Mutagenesis Protocol and the CRIMP Toolkit. Miles LB, Calcinotto V, Sonntag C, Lee C, Bryson-Richardson RJ. bioRxiv. July 14, 2023; doi: 10.1101/2023.07.13.548929 .
Description
The CRIMPkit contains 30 ready-to-use plasmid vectors for easy and complete mutagenesis in any reading frame without requiring custom sequences, modification, or subcloning.
To make targeted insertions, CRISPR/Cas9 is used both to generate a double-stranded break in an intron or 5'UTR of a target gene and to linearize the targeting plasmid. Insertion is mediated by non-homologous end-joining (NHEJ) repair. Once inserted, a fluorescent reporter is expressed under the control of the targeted gene's promoter. This insertion disrupts native gene expression by inducing complete transcriptional termination, producing loss of function mutants without genetic compensation. The insertion also provides a readout of the targeted gene's expression pattern and enables visual identification of successfully mutagenized fish. Vectors are available in all three reading frames after the splice acceptor, enabling this toolkit to target any intron of any target gene.
Although the plasmids in this toolkit are codon-optimized for use in zebrafish, the approach and toolkit could be used in any species.
- mTagBFP2 expression replaces native alpha-actin (Actc1b) and labels muscle cells in the targeted insertion mutant line Ti(actc1bint2-mTagBFP2). This line was generated by a targeted insertion of pSA_0_mTagBFP2 plasmid into intron-2 of actc1b. mTagBFP2 is false-colored magenta. Ventral view of the zebrafish head at 4 dpf. Field of view: 750 μm.
Kit Documentation
Protocols and documentation (PDF, 463 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 CRIMPkit was a gift from Robert Bryson-Richardson (Addgene kit #1000000225).”
For your Reference section:
CRIMP: A CRISPR/Cas9 Insertional Mutagenesis Protocol and the CRIMP Toolkit. Miles LB, Calcinotto V, Sonntag C, Lee C, Bryson-Richardson RJ. bioRxiv. July 14, 2023; doi: 10.1101/2023.07.13.548929 .
CRIMPkit - #1000000225
- 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 |