targeting microrna pathways in the intestinal stem cell
 by multiplex somatic mutagenesis

I propose to study how the Let-7 family of miRNAs represses colorectal tumorigenesis. I have compelling data that loss of this family of tumor suppressor miRNAs (a type of small RNA that inhibits specific genes or “targets”) drives tumor initiation and progression in the intestine. I will study this miRNA pathway through the use of CRISPR/Cas and a novel Piggybac DNA transposon for targeted somatic mutagenesis in novel mouse models. These new technologies allow one to target a large number of genes and our efforts represent the first efforts to do so, in vivo.

My project will involve novel transgenic models using  two systems: the CRISPR/Cas system for genome editing (for turning genes off) and the Piggybac DNA transposon for conditional transgenesis (to turn genes on). I have compelling plans for use of these systems for functional genomics to study stem cell dynamics and tumorigenesis using the mouse intestine as a model system. I have generated transgenic mice expressing an intestine- specific and inducible Cas9D10A nuclease (iiCas9N mice) for targeted somatic mutagenesis in the intestine, which I plan to use as a platform for somatic mutagenesis of tumor-suppressor miRNA families, starting with the Let-7 miRNA family. Second, I am implementing a novel application of the Piggybac DNA transposon for conditional expression of multiple transgenes to test the effects of Let-7 target mRNAs on stem cells and cancer. These approaches enable us to study the large Let-7 gene family (that may function cooperatively among all 12 Let-7 genes) and the combinatorial interactions of Let-7 targets that are turned on following Let-7 mutation or depletion.