21:T466,Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAF^V600E. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAF^V600E. Adult zebrafish expressing BRAF^V600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAF^V600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAF^V600E-mediated transformation.22:T428,CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

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