In vivo tracing of the Cytokeratin 14 lineages using self-cleaving guide RNAs and CRISPR/Cas9.

The current gold-standard for genetic lineage tracing in transgenic mice is based on cell-type specific expression of Cre recombinase. As an alternative, we developed a cell-type specific CRISPR/spCas9 system for lineage tracing. This method relies on RNA polymerase II promoter driven self-cleaving guide RNAs (scgRNA) to achieve tissue-specificity. To demonstrate proof-of-principle for this approach a transgenic mouse was generated harbouring a knock-in of a scgRNA into the Cytokeratin 14 (Krt14) locus. Krt14 expression marks the stem cells of squamous epithelium in the skin and oral mucosa. The scgRNA targets a Stop cassette preceding a fluorescent reporter in the Ai9-tdtomato mouse. Ai9-tdtomato reporter mice harbouring this allele along with a spCas9 transgene demonstrated precise marking of the Krt14 lineage. We conclude that RNA polymerase II promoter driven scgRNAs enable the use of CRISPR/spCas9 for genetic lineage tracing.

Development of a Portable LAMP Assay for Detection of Neofabraea perennans in Commercial Apple Fruit.

Bull's eye rot (BER) is a major economic postharvest disease of apple and pear that can be caused by four Neofabraea species: N. perennans, N. alba, N. malicorticis, and N. kienholzii. In Central Washington, BER is predominantly caused by N. perennans. The fungus infects fruit preharvest, and because of the dry growing season in the region, infections remain latent with symptoms expressed only after 3 to 4 months of storage, when BER incidences as high as 20% can been seen, especially in rainy seasons and on susceptible cultivars. To ensure early and efficient infection detections before BER symptoms become visible at point-of-care locations, a portable diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using the ß-tubulin gene. The LAMP assay was optimized and tested for specificity and sensitivity using DNA extracted from pure cultures of N. perennans and seven other fungal species. The results showed that the selected LAMP primer set was specific to N. perennans and highly sensitive as it detected DNA concentrations as low as 0.001 ng/µl after only 10 min. The assay was validated for N. perennans detection on artificially inoculated apples using a portable thermocycler, Genie II, without the need for DNA extraction. The LAMP assay detected N. perennans on apples inoculated with spore suspensions 3 weeks prior to harvest at concentrations of 103 spores/ml or higher. The assay was further validated using commercial Piñata apples from organic and conventional orchards, demonstrating the ability of this technique to amplify N. perennans from asymptomatic fruit in a commercial setting 3 months before commercial maturity. The LAMP assay developed for N. perennans detection can be easily expanded to detect the other BER causal species. LAMP has potential to be used in orchards and at point-of-care facilities to better inform on BER management at different fruit growth stages, and it has potential to be utilized to better understand the epidemiology of Neofabraea spp.

Strong ubiquitous micro-promoters for recombinant adeno-associated viral vectors.

Significant progress has been made in developing recombinant adeno-associated virus (rAAV) for clinical gene therapy. While rAAV is a versatile gene delivery platform, its packaging limit of 4.7 kb limits the diseases it can target. Here, we report two unusually small promoters that enable the expression of larger transgenes than standard promoters. These micro-promoters are only 84 (MP-84) and 135 bp (MP-135) in size but have activity in most cells and tissues comparable to the CAG promoter, the strongest ubiquitous promoter to date. MP-84- and MP-135-based rAAV constructs displayed robust activity in cultured cells from the three different germ-layer lineages. In addition, reporter gene expression was documented in human primary hepatocytes and pancreatic islets and in multiple mouse tissues in vivo, including brain and skeletal muscle. MP-84 and MP-135 will enable the therapeutic expression of transgenes currently too large for rAAV vectors.

Hidden Host Mortality from an Introduced Parasitoid: Conventional and Molecular Evaluation of Non-Target Risk.

Hidden trophic interactions are important in understanding food web ecology and evaluating the ecological risks and benefits associated with the introduction of exotic natural enemies in classical biological control programs. Although non-target risk is typically evaluated based on evidence of successful parasitism, parasitoid-induced host mortality not resulting in visible evidence of parasitism (i.e., nonreproductive effects) is often overlooked. The adventive establishment of Trissolcus japonicus, an exotic parasitoid of the introduced stink bug Halyomorpha halys, provides an opportunity to investigate the total impact of this parasitoid on target and non-target hosts in the field. We developed a new methodology to measure nonreproductive effects in this system, involving a species-specific diagnostic PCR assay for T. japonicus. We applied this methodology to field-deployed eggs of four pentatomid species, coupled with traditional rearing techniques. Nonreproductive effects were responsible for the mortality of an additional 5.6% of H. halys eggs due to T. japonicus, and were even more substantial in some of the non-target species (5.4-43.2%). The observed hidden mortality of native non-target species from an introduced parasitoid could change predictions about direct and indirect ecological interactions and the efficacy of biological control of the target pest.