CRISPR/Cas9 Gene Targeting by Pronuclear Injection
Gene knock-out using CRISPR technology
To obtain gene knock-out mice using CRISPR technology, synthetic, modified sgRNAs or crRNA/trRNAs, provided by the investigator, will be electroporated with Cas9 protein into pronuclear-stage embryos. Surviving embryos will be transferred into the oviduct of foster females. All pups that result from the procedure are potentially mosaic for knock-out mutations via indel formation. Pups will be transferred to the investigator for analysis and subsequent mating to the F1 generation to establish the mutation in the germline.
Service Requirements, Guarantees and Fees
- sgRNA or crRNA/trRNA must be obtained from an approved vendor and have modified ends. Please contact the GEMF co-director for suggestions. A minimum of 2 µg of sgRNA is required (1.5 nmol is sufficient).
- Two electroporation days will be scheduled for CRISPR/Cas9 knock-out projects.
- We cannot guarantee correct targeting.
- Due to patent regulations, CRISPR injections may currently only be provided for MD Anderson investigators or UT investigators at institutions that lack gene modification services.
- Fee for knock-out projects: $1,523 for MD Anderson investigators, $2,378 for other UT investigators
- Additional days (upon request): $800/day, $1248 for other UT investigators
- A single electroporation day may be purchased in lieu of a 2-day procedure. However, we cannot guarantee that all contributing procedures will be optimal: $800/day, $1248 for other UT investigators
- 10 injected blastocysts may be requested for sgRNA analysis: $84 for MD Anderson investigators, $131 for other UT investigators.
Gene knock-in using CRISPR technology
To obtain gene knock-in mice using CRISPR technology, the investigator will need to submit DNA (for homology driven repair) and sgRNAs or crRNA/trRNAs. These materials will be mixed with Cas9 protein and injected into pronuclear-stage embryos. Surviving embryos will be transferred into the oviduct of foster females. All pups that result from the procedure are potentially mosaic for various mutations, including knock-out mutations that occur via indel formation, knock-in mutations containing some or all of the DNA supplied, or a combination of all of these. Pups will be transferred to the investigator for analysis and subsequent mating to the F1 generation to establish the mutation in the germline.
Service Requirements, Guarantees and Fees
- synthetic sgRNA or crRNA/trRNA must be obtained from an approved vendor. Please contact the GEMF co-director for suggestions. A minimum of 2 µg of sgRNA is required.
- DNA for hdr should be supplied as a supercoiled, double-stranded plasmid or ss oligonucleotide donor. A minimum of 3µg is required if purified. For purification by the GEMF, a minimum of 30 µg is required. ALL DNA MUST BE ISOLATED USING ENDOTOXIN-FREE PROCEDURES. Please consult with the GEMF co-director for assistance in determining the best DNA donor to achieve your goals.
- Three injection days will be scheduled for CRISPR/Cas9 knock-in projects.
- We cannot guarantee correct targeting.
- Due to patent regulations, CRISPR injections may currently only be provided for MD Anderson investigators or UT investigators at institutions that lack gene modification services.
- Fee for knock-in injections: $2,310 for MD Anderson investigators, $3,607 for other UT investigators.
- Additional injection days (upon request): $800/day. $1248 for other UT investigators
- 10 injected blastocysts may be requested for sgRNA analysis: $84 for MD Anderson investigators, $131 for other UT investigators from UT institutions that lack gene modification services.