CRISPR

Background:

CRISPR/Cas9 is a powerful genome-editing tool that is redefining the boundaries of biological research. The GeTT at Roswell Park Comprehensive Cancer Center is pleased to introduce a FULL CRISPR-based gene-editing platform to modify your mouse genome by global knockouts, small amino acid substitutions or other small tag knockins as well as larger gene fusions and conditional allele knockins. We have had over 60 projects since 2013, and since 2017 with the inception of easi-CRISPR, we have had success in 99% of our projects.

Design and production of your guide RNA and oligos:

The GeTT will work with you to design the reagents needed for a successful project or design the entire project for you.

CRISPR Electroporation used for KNOCKOUTs and small DNA insertions (base changes, amino acid substitutions (≤200bp)):

The GETT will harvest and inject fertilized oocytes with CRISPR reagents. RNAs and protein (sometimes DNA as well) are concurrently electroporated and handled with the utmost care to prevent degradation. Targeting DNA with homologous arms can be co-injected to direct integration/homologous recombination to that site. DNA and RNA are prepared and purified by the GeTT. Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA, and will also be identified by an ear tag.

CRISPR One-cell Injection (typically used for larger DNA insertions (>500bp):

The GETT will harvest and inject fertilized oocytes with CRISPR reagents. RNAs and protein (sometimes DNA as well) are concurrently injected and handled with the utmost care to prevent degradation.

Targeting DNA with homologous arms can be co-injected to direct integration/homologous recombination to that site. DNA and RNA are prepared and purified by the GeTT. Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA, and will also be identified by an ear tag.

CRISPR Two-cell Injection (typically used for larger DNA insertions (>500bp):

The GETT will harvest and inject fertilized oocytes with CRISPR reagents. RNAs and protein (sometimes DNA as well) are concurrently injected and handled with the utmost care to prevent degradation.

Targeting DNA with homologous arms can be co-injected to direct integration/homologous recombination to that site. DNA and RNA are prepared and purified by the GeTT. Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA, and will also be identified by an ear tag.

According to a recent publication by the Rossant lab: they were able to achieve a more-than-tenfold increase in knock-in efficiency over standard methods using 2-cell injection. (Gu et al. Nature Biotechnology volume 36, pages 632–637 (2018)).

The GeTT highly suggests getting targeted NGS done on the founder and F1 animals resulting from CRISPR injections/electroporations, as mosaicism is always a concern.