SUMMARY: Murine Mutagenesis Core In recent years neuroscience research has experienced many transformative technical advances. Mouse genetics is a major contributor to this technological revolution. NINDS-supported investigators at JHU SOM rely heavily on mutant and transgenic mouse lines to study the nervous system, both in health and disease. However, the generation of these transgenic mouse lines, which in many cases are tailor-made, expensive, and time-consuming to produce, requires extensive dedicated effort and equipment. Moreover, to achieve success using these transgenic technologies, users must have considerable technical knowledge. The goal of the Murine Mutagenesis Core (MMC) is to lower barriers for generating mouse mutants by providing a unique suite of mouse mutagenesis services for neuroscience investigators at JHU SOM. The MMC offers NINDS- supported investigators, and other scientists engaged in research consistent with the NINDS mission, access to state-of-the-art techniques, including: homologous recombination-mediated targeting in mouse ES cells, karyotyping of ES cells, cryopreservation of crucial mouse lines, and the use of targeted ES cells from commercial and noncommercial sources. Importantly, the highly experienced MMC personnel provide face-to- face advice on targeting construct design, the use of recombineering technology to make these constructs, project trouble-shooting as work progresses, and technical feedback which is lacking in cores outside of JHU. Owing to recent success by the MMC in employing CRISPR, ZFN, and integrase based mutagenesis and gene targeting, the MMC proposes expanding its services to utilize these non-ES cell based technologies that have significantly shortened the timeline for generating mutant mouse alleles and have reduced the cost for investigators compared with traditional homologous recombination approaches. The proposed new services (many of which are not commercially available) include: 1) CRISPR induced small deletion mutations; 2) CRISPR-introduced floxed conditional and epitope-tagged alleles; 3) generation of large insertions using the CRISPR approach; 4) CRISPR-induced additional mutations in existing transgenic lines; and 5) introduction of useful genes, such as those encoding various forms of GCaMPs and DREADDs, into the Rosa26 locus by integrase-based integration. Since there are many challenges in implementing the evolving CRISPR technologies, and since there is still value in using targeted ES cells from commercial sources, the MMC ES work will be maintained. Given its success and productivity during the past funding period, the MMC proposes continuing to provide Primary Center Investigators and other neuroscientists at JHU with the opportunity to generate innovative mouse lines to study basic mechanisms of nervous system function and the causes of neurologic diseases. In addition, the MMC proposes continuing to encourage collaboration among neuroscientists at JHU and facilitating the utilization of state-of-the-art techniques to accelerate advances in neuroscience research.