CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

To monitor rodent colony health in research facilities, soiled-bedding sentinel (SBS) animals have traditionally been used. SBS can be tested by various methods, which may include serology, PCR analysis, and necropsy. Several pathogens are unreliably detected by using SBS or transmitted poorly through soiled bedding, and collection and evaluation of SBS samples can be time-intensive. Recently, exhaust air dust (EAD) testing through PCR analysis has emerged as an adjunct or replacement method for rodent colony health monitoring. EAD monitoring may provide a more efficient, sensitive, and humane method for monitoring health status. Using both EAD and SBS health monitoring, we evaluated colony health over the course of 1 y in 3 research barrier rooms in which mice were housed exclusively on IVC racks. Three pathogens—Helicobacter spp., Rodentibacter spp. (previously Pasteurella pneumotropica), and murine norovirus (MNV)—were not excluded in 2 of the rooms, and we expected that these mice would test positive with some regularity. EAD monitoring was significantly more sensitive than SBS for detection of the bacterial agents. SBS failed to detect Helicobacter spp. at time points when EAD had 100% detection in the rooms that did not exclude the bacteria. The detection of MNV did not differ between health monitoring systems at any time point. The findings suggest that EAD is especially valuable in detecting bacteria poorly transmitted through soiled bedding. In addition, the corresponding results with MNV detection suggest that EAD surveillance can reliably be implemented as an alternative to SBS monitoring in a facility in which mice are housed exclusively on IVC racks.

Rodent vivaria have traditionally used soiled bedding sentinel (SBS) health-monitoring programs to detect and exclude adventitious pathogens that could affect research results. Given the limitations of SBS, a likely reduction in animal usage, and a decrease in animal care staff labor, exhaust air dust (EAD) health monitoring has been evaluated by several groups for its efficacy in detecting pathogens when used as a complete replacement for traditional SBS health-monitoring programs. Compared with SBS, EAD has also been shown to provide increased sensitivity for the detection of multiple pathogens. After implementing EAD at our institution, we conducted an analysis to compare the annual costs of the 2 health-monitoringprograms. The EAD program was found to be 26% less expensive than SBS. In addition to these cost savings, EAD decreased the amount of time spent by the staff on heath-monitoring activities. For veterinary technicians, this decrease in time was calculated as a savings of 150 h annually, almost 3 h each week. Finally, the EAD program replaced the use of live sentinel animals, decreasing the associated yearly usage from 1,676 animals to zero.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We compared synthetic, chemically modified sgRNAs to in vitro transcribed sgRNAs and demonstrate the increased activity of synthetic sgRNAs in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to optimize the design of synthetic DNA templates to promote HDR. Utilizing these principles, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic sgRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

Lactate dehydrogenase elevating virus (LDV) continues to be one of the most common contaminants of cells and cell byproducts. As such, many institutions require that tumor cell lines, blood products, and products derived or passaged in rodent tissues are free of LDV as well as other pathogens that are on institutional exclusion lists prior to their use in rodents. LDV is difficult to detect by using a live-animal sentinel health monitoring program because the virus does not reliably pass to sentinel animals. After switching to an exhaust air dust health monitoring system, our animal resources center was able to detect a presumably long-standing LDV infection in a mouse colony. This health monitoring system uses IVC rack exhaust air dust collection media in conjunction with PCR analysis. Ultimately, the source of the contamination was identified as multiple LDV-positive patient-derived xenografts and multiple LDV-positive breeding animals. This case study is the first to demonstrate the use of environmental PCR testing as a method for detecting LDV infection in a mouse vivarium.