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Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

ERDC/EL TR-18-1 Environmental Quality/Installations (EQ/I) Development and Characterization of Small-Scale Washing Systems for Removal of Depleted Uranium Oxides Environmental Laboratory Ronald J. Unz, Jay P. McCown, Charles A. Waggoner, Steven L. Larson, and John H. Ballard January 2018 Approved for public release; distribution is unlimited. The U.S. Army Engineer Research and Development Center (ERDC) solves the nation’s toughest engineering and environmental challenges. ERDC develops innovative solutions in civil and military engineering, geospatial sciences, water resources, and environmental sciences for the Army, the Department of Defense, civilian agencies, and our nation’s public good. Find out more at www.erdc.usace.army.mil. To search for other technical reports published by ERDC, visit the ERDC online library at http://acwc.sdp.sirsi.net/client/default.Environmental Quality/Installations (EQ/I) ERDC/EL TR-18-1 January 2018 Development and Characterization of Small-Scale Washing Systems for Removal of Depleted Uranium Oxides Ronald J. Unz, Jay P. McCown, and Charles M. Waggoner Institute for Clean Energy Technology, Mississippi State University 205 Research Blvd. Starkville, MS 39702 Steven L. Larson and John H. Ballard Environmental Laboratory U.S. Army Engineer Research and Development Center 3909 Halls Ferry Rd. Vicksburg, MS 39180 Final Report Approved for public release; distribution is unlimited. Prepared for Headquarters, U.S. Army Corps of Engineers Washington, DC 20314-1000 Under Department of Defense Appropriations Act Program Element 0603728A, “Depleted Uranium (DU) Clearance from DOD Ranges” ERDC/EL TR-18-1 iv Abstract Researchers from the Mississippi State University Institute for Clean En-ergy Technology (MSU-ICET) and the U.S. Army Engineer Research and Development Center (ERDC) have identified procedures and methodolo-gies for identifying leaching solutions to assist in the removal of depleted uranium (DU) oxides from contaminated soils. Th

ERDC/EL TR-15-4 Development of a Scalable Process Control System for Chemical Soil Washing to Remove Uranyl Oxide Environmental Laboratory Jay P. McCown, Ronald J. Unz, Charles A. Waggoner, John H. Ballard, Steven L. Larson, and Per Arienti May 2015 Approved for public release; distribution is unlimited. The US Army Engineer Research and Development Center (ERDC) solves the nation’s toughest engineering and environmental challenges. ERDC develops innovative solutions in civil and military engineering, geospatial sciences, water resources, and environmental sciences for the Army, the Department of Defense, civilian agencies, and our nation’s public good. Find out more at www.erdc.usace.army.mil. To search for other technical reports published by ERDC, visit the ERDC online library at http://acwc.sdp.sirsi.net/client/default.ERDC/EL TR-15-4 May 2015 Development of a Scalable Process Control System for Chemical Soil Washing to Remove Uranyl Oxide Jay P. McCown, Ronald J. Unz, and Charles A. Waggoner Institute for Clean Energy Technology Bagley College of Engineering Mississippi State University, MS 39759 Steven L. Larson and John H. Ballard Environmental Laboratory U.S. Army Engineer Research and Development Center 3909 Halls Ferry Rd. Vicksburg, MS 39180-6199 Per Arienti U.S. Army Armament Research and Development Engineering Center Picatinny Arsenal, NJ 07806 Final report Approved for public release; distribution is unlimited. Prepared for U.S. Army Corps of Engineers Washington, DC 20314-1000 ERDC/EL TR-15-4 ii Abstract The U.S. Army has responsibility for maintaining or managing a large number of facilities that are or have been used for training troops and developing/testing equipment and munitions, including ranges that may have been contaminated with uranium. Licenses issued by the Nuclear Regulatory Commission (NRC) for use of radiological materials such as depleted uranium (DU) specify the isotopes that can be used, along with possession limits for the site. U

The United States (US) Army and the other services are mandated to comply with all federal, state, and local environmental regulations. Recent concerns over potential human exposure to depleted uranium (DU) at US Army test ranges, primarily, have resulted in research into numerous innovative remediation technologies. Developing methods and processes for estimating the life cycle costs (LCC) of implementing these various techniques is important in identifying cost-effective solutions. Total ownership costs (TOC) models were developed for four candidate alternatives using two new technologies; Alternative 1- Physical Separation and Chemical Treatment; Alternative 2- Selective Excavation; Alternative 3- Selective Excavation with Physical Separation and Chemical Treatment; Alternative 4- Containment and Monitoring. The team chose a generic sandy soil site roughly 10 miles square for cost estimates. A bottom up estimate was applied to all alternatives in order to get a baseline cost; Alternative 3 had the best estimate for an efficient and effective remediation method. A cost estimating relationship was generated and simulation-based costing (SBC) was then applied to Alternative 3. Lastly, two alternatives were evaluated for DU remediation of catchboxes. Identifying the key cost drivers from SBC modeling is significant to future investments in research and development., The original document contains color images. Prepared in collaboration with Applied Research Associates, Inc, Vicksburg, MS.

Similarly to other cytoplasmic genetic elements, intracellular endosymbionts of animals are passed to the next generation of hosts through the egg. This mode of transmission is key to understanding the evolution of these symbiotic associations. For instance, maternal inheritance creates a sexual asymmetry, as genetic variants that are advantageous to females would persist even when harmful to males. Wolbachia are well known for using such sexual asymmetry to their advantage: by actively reducing the reproductive chances of non-infective hosts, they often increase the relative fitness of infected matrilines. However, so long as these genetic elements prevent the normal transmission of host genetic material to the next generation, there would be antagonistic selective pressure over the reproductive phenotype from the host. This means that Wolbachia-related parasitic phenotypes, similarly to sex-antagonist mitochondrial mutations, would be likely attenuated by compensatory adaptation.Despite being often perceived as parasites, emerging evidence on beneficial effects of Wolbachia offers new perspectives on the nature of Wolbachia-host interactions. Similarly to the current understanding on the maintenance of functional mitochondrial genetic variation, Wolbachia that pose advantages contingent to the host nuclear background and the environment may be selectively favoured and maintained at equilibrium, even in the face of attenuation of reproductive manipulation phenotypes. This thesis explores various aspects of the interaction between cytoplasmic (namely Wolbachia and mitochondria) and nuclear genomes using Drosophila flies as models. Additionally to their well-characterised mitochondrial genomes, Drosophila can serve as host to a variety of Wolbachia infections with markedly distinct phenotypes, which provides the opportunity to test hypothesis regarding host adaptation to phenotypic effects of cytoplasmic variants. Three major conclusions can be drawn from this thesis