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Background & Aims: Assessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or peri-zygotic embryos. These approaches are time consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a Rapid In vivo Multiplexed Editing (RIME), and to provide a proof-of-concept of this system.Approach & Results: RIME, a system wherein CRISPR/Cas9 technology, paired with adenoassociated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver.The method is quick, requiring as little as 1 month from conceptualization to knockout (KO), and highly efficient, enabling editing in >95% of target cells. To highlight its utility, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation. Conclusion:RIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo..

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate ″reprogramming″ by opening new chromatin sites for expression that can attract transcription factors from the starting cell′s enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.