GeneWeld Vectors for Targeted Integration Using CRISPR/Cas9
(Kit #
1000000154
)
Depositing Lab: Jeffrey Essner
Vectors for integration using short homology and CRISPR/Cas9. The vectors provide the ability and flexibility to produce knockout alleles with and without a fluorescent reporter, fusion proteins with endogenous genes, and Cre drivers.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
GeneWeld: a method for efficient targeted integration directed by short homology Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Torrie ME, Weiss TJ, Vollbrecht MK, Lan M, McKeighan KC, Levey J, Ming Z, Wehmeier A, Mikelson CS, Haltom JA, Kwan KM, Chien C, Balciunas D, Ekker SC, Clark KJ, Webber BR, Moriarity B, Solin SL, Carlson DF, Dobbs DL, McGrail M, Essner JJ. bioRxiv. 2019 May 25; doi: 10.1101/431627. bioRxiv preprint.
Description
The GeneWeld Kit can be used for targeted integration in human cells, zebrafish embryos, and pig fibroblasts. GeneWeld uses the CRISPR/Cas9 system, along with a donor vector, to create knock-ins including fluorescent reporters, fusion proteins, and transcriptional regulators. Donor vectors with as little as 24 to 48 base pairs of homology can be used to direct knock-in sequences to a genomic loci of interest.
To generate a knock-in using CRISPR/Cas9, Cas9 is used to generate a double-stranded DNA break at three locations: the genomic loci of interest, and on either side of the homology arms within the donor vector. This is accomplished using an sgRNA targeting the knock-in site, as well as a universal sgRNA targeting the donor vector sequence. This universal sgRNA is not predicted to target any sites in human, pig, or zebrafish genomic DNA. The excised homology arms on either side of the donor plasmid cargo are complementary to the DNA on each side of the double-stranded break at the genomic loci of interest. This initiates homology-mediated end joining, resulting in a knock-in at the locus of interest.
A detailed protocol describing this methodology can be found under Protocols & Resources.
Targeting of the vim1 gene in zebrafish with pPRISM-Fuse produces a fusion protein between the vim1 protein and mRFP. Lateral view of the zebrafish trunk at 3 days post fertilization. mRFP expression is color coded for different confocal planes.
Kit Documentation
GeneWeld Protocol 575.0Â 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 GeneWeld Vectors for Targeted Integration Using CRISPR/Cas9 was a gift from Jeffrey Essner (Addgene kit #1000000154).”
For your Reference section:
GeneWeld: a method for efficient targeted integration directed by short homology Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Torrie ME, Weiss TJ, Vollbrecht MK, Lan M, McKeighan KC, Levey J, Ming Z, Wehmeier A, Mikelson CS, Haltom JA, Kwan KM, Chien C, Balciunas D, Ekker SC, Clark KJ, Webber BR, Moriarity B, Solin SL, Carlson DF, Dobbs DL, McGrail M, Essner JJ. bioRxiv. 2019 May 25; doi: 10.1101/431627. bioRxiv preprint.
GeneWeld Vectors for Targeted Integration Using CRISPR/Cas9 - #1000000154
- 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
| Kanamycin |