PROJECT SUMMARY/ABSTRACT
The long-term goal of this project is to increase the ability of researchers to create faithful mouse and stem-cell
models of human cancers and other diseases. Currently available genetic-engineering approaches, including
the CRISPR-Cas9 system, which has revolutionized genome editing, lack the capacity for efficient integration of
large DNA constructs (> 10 kilobases; kb) in mouse zygotes and mouse and human stem cells. This limitation
significantly hinders the modeling of human diseases, including cancer. For example, tandem duplications (TDs),
super-enhancers (SEs; large clusters of transcriptional enhancers), and large non-coding structural variants have
been linked to human diseases, including cancers, but available technologies do not permit modeling such large
variants in whole animals or cell lines. To fill this gap, we will develop a gene-editing toolbox that couples the
precision of the CRISPR-Cas9 system with the fidelity and efficiency of the serine integrase Bxb1 to enable
rapid, efficient insertion of large DNA constructs in mice, mouse embryonic stem cells, and human induced
pluripotent stem cells (hiPSCs). Bxb1 integrase uses DNA attachment sites (attP in the genome, attB in the
donor DNA) as substrates for catalyzing efficient transgenesis. We show that our innovative Cas9-Bxb1 toolbox
can precisely integrate DNA constructs up to ~43 kb in length in mice. Here, in three aims we will further develop
and validate the toolbox to enable precise transgenesis of large DNA constructs (~100 kb) and to facilitate
generation of DNA rearrangements. Aim 1: Optimize the Cas9-Bxb1 toolbox for insertion of large DNA (10 to
100 kb) constructs into mouse zygotes. We will use reporter constructs with differing lengths to determine the
maximum length of DNA construct that can be inserted efficiently, and will validate a one-step protocol for rapid
generation of transgenic mice without the need to first generate and characterize mice with attachment sites.
Aim 2: Generate mouse and hiPSC models of human diseases, including cancer, using the Cas9-Bxb1 toolbox.
Using our toolbox to insert large genomic variants, we will generate a mouse model of breast cancer (insertion
of a 23.7-kb TD), hiPSC model of triple negative breast cancer (27.2-kb SE), and mouse model of Hirschsprung
disease (~80-kb human risk allele). Aim 3: Enable use of the Cas9-Bxb1 toolbox for generation of DNA
rearrangements. In cre-lox recombination systems, cre catalyzes recombination between two loxP sites flanking
a target locus, enabling diverse DNA rearrangements. Studies suggest that cre-recombination efficacy is limited
by the inter-loxP-site distance and the particular genomic site targeted. We will determine whether the Cas9-
Bxb1 toolbox is more efficient than cre-lox for generation of DNA rearrangements, by determining Bxb1-mediated
recombination efficacy at different inter-attP/attB distances. Successful completion of this project will provide the
community with three new models for future studies, and a versatile tool for development of novel and improved
mouse and hiPSC models of cancer and other diseases.
