Poster - #VH-54903 - Keywords: Bxb1 RMCE dPCR CRISPR mESC JAX

Powerful New Technologies Combine to Improve Efficient Generation of Precision Mouse Models

Benjamin E. Low, Whitney Martin, Ethan Saville, Kathy J. Snow, Michael V. Wiles, Stephen A. Murray
The Jackson Laboratory, Bar Harbor, Maine 04609 USA

ABSTRACT:
Introduction
With our ever-expanding knowledge of the genetic causes of disease there is an increased need for advanced, precision animal models to understand disease pathobiology and to serve as platforms for preclinical investigation.

Aims
To this end, we have established a robust, streamlined platform for the precision engineering of disease-causing mutations coupled with humanization of the mouse genome. This platform allows for large-scale modification of the mouse genome in both mouse embryonic stem cells (mESCs) and mouse zygotes and takes advantage of powerful technologies including CRISPR-Cas9 and Bxb1 Recombinase Mediated Cassette Exchange (B-RMCE).

Methods
CRISPR-assisted gene targeting is used to pre-position a Bxb1 Landing Pad allele into any locus on a defined mouse strain. Delivery of a donor plasmid and the Bxb1 Integrase mRNA enables RMCE of the cassette at high efficiency, excluding the vector backbone by design. The result is a single-copy precision transgenic allele positioned in a defined genetic locus. Digital PCR enables rapid identification of founder candidates, including low contribution mosaics that might otherwise be missed by standard screening techniques. Copy number verification in the line(s) selected for breeding prevents downstream complications from undesired off-target insertion events. Long-Read Sequencing by ONT ensures that the allele is completely defined, down to the single-base level.

Results
Using this technique, we have created many new mouse lines with genetic modifications ranging from 1.5kb to 77.3kb, with an average success rate of 15% for RMCE-positive founders. Our ongoing work with mESCs is showing promising early results for BAC-scale replacements.

Conclusions
Combined, these techniques enable efficient production and verification of the designed allele with confidence in a streamlined, rapid manner.