Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans.

Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here, we describe a modular safe-harbor transgene insertion (MosTI) for use in Caenorhabditis elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by nonhomologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25-75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7-44%) at modular safe-harbor transgene insertion landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for 8 integrated arrays (6-37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of 1 array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.

Systemic Design Approach to a Real-Time Healthcare Monitoring System: Reducing Unplanned Hospital Readmissions.

Following hospital discharge, millions of patients continue to recover outside formal healthcare organizations (HCOs) in designated transitional care periods (TCPs). Unplanned hospital readmissions of patients during TCPs adversely affects the quality and cost of care. In order to reduce the rates of unplanned hospital readmissions, we propose a real-time patient-centric system, built around applications, to assist discharged patients in remaining at home or in the workplace while being supported by care providers. Discrete-event system modeling techniques and supervisory control theory play fundamental roles in the system's design. Simulation results and analysis show that the proposed system can be effective in documenting a patient's condition and health-related behaviors. Most importantly, the system tackles the problem of unplanned hospital readmissions by supporting discharged patients at a lower cost via home/workplace monitoring without sacrificing the quality of care.