Wierson, W. A. | Search bioRxiv

bioRxiv2018

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

genetics↗

bioRxiv2018

Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

Retinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity correlates with failure of neural precursors to initiate quiescence and transition to differentiation. rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current interest in Rbbp4 as a potential druggable target.\n\nAuthor SummaryExamining the developmental mechanisms controlling neural stem and progenitor cell behavior is critical to our understanding of the processes driving brain tumor oncogenesis. Chromatin remodelers and their associated adaptor proteins are thought to be key drivers of brain development and disease through epigenetic regulation of gene expression and maintenance of genome integrity, but knowledge of their in vivo roles in vertebrate neurogenesis is limited. The chromatin remodeler adaptor protein Rbbp4 has recently been shown to function in a mouse model of neuroblastoma and in glioblastoma multiforme cell resistance to the chemotherapeutic temozolomide. However, an in vivo requirement for Rbbp4 in neurogenesis has only just been shown by isolation of a recessive lethal mutation in zebrafish rbbp4. Here we provide conclusive genetic evidence that zebrafish rbbp4 is essential in neural stem and progenitor cell function during development. Our data reveal for the first time in vivo that Rbbp4 prevents DNA damage and activation of Tp53 signaling pathway that leads to programmed cell death. Importantly, neural progenitors that are mutant for the tumor suppressor Rb1 also depend on Rbbp4 for survival. Finally, we show that neural stem cells that have lost Rbbp4 cease dividing, and may enter a senescent like state. Together, these observations provide novel evidence that elevated expression of Rbbp4 in rb1-mutant tumors may contribute to cancer cell survival by blocking senescence and/or DNA damage-induced cell death.

genetics↗