The contribution of homology arms to nuclease-assisted genome engineering.

Designer nucleases like CRISPR/Cas9 enable fluent site-directed damage or small mutations in many genomes. Strategies for their use to achieve more complex tasks like regional exchanges for gene humanization or the establishment of conditional alleles are still emerging. To optimize Cas9-assisted targeting, we measured the relationship between targeting frequency and homology length in targeting constructs using a hypoxanthine-guanine phosphoribosyl-transferase assay in mouse embryonic stem cells. Targeting frequency with supercoiled plasmids improved steeply up to 2 kb total homology and continued to increase with even longer homology arms, thereby implying that Cas9-assisted targeting efficiencies can be improved using homology arms of 1 kb or greater. To humanize the Kmt2d gene, we built a hybrid mouse/human targeting construct in a bacterial artificial chromosome by recombineering. To simplify the possible outcomes, we employed a single Cas9 cleavage strategy and best achieved the intended 42 kb regional exchange with a targeting construct including a very long homology arm to recombine ∼42 kb away from the cleavage site. We recommend the use of long homology arm targeting constructs for accurate and efficient complex genome engineering, particularly when combined with the simplifying advantages of using just one Cas9 cleavage at the genome target site.

The role of environmental factors on the evolution of phenotypic diversity in vesicular stomatitis virus populations.

Virus adaptation to an ever-changing environment requires the availability of variants with phenotypes that can fulfil new requirements for replication. High mutation rates result in the generation of these variants. The factors that contribute to the maintenance or elimination of this diversity, however, are not fully understood. This study used a collection of vesicular stomatitis virus strains generated under different conditions to measure the extent of variation within each population, and tested the effects of several environmental factors on diversity. It was found that the host-cell type used for selection sometimes had an effect on the extent of variation and that there may be different levels of variation over time. Persistent infections promoted higher levels of diversity than acute infections, presumably due to complementation. In contrast, environmental heterogeneity, host breadth and the cell type used for testing (as opposed to the cell type used for selection) did not seem to have an effect on the amount of phenotypic diversity observed.

P18-A148†Assymetrical injector for endothelium-in DMEK without the need of pull-through technique.

PURPOSE: One of latest surgical development of preloaded Descemet membrane endothelial keratoplasty (DMEK) is the delivery of the graft with the endothelium inwards, which allows for a very fast operation, but requires a pull-through surgical technique. Although the tri-folded, endo-in DMEK technique has significant advantages, the absence of proper surgical instruments that could allow their use without the 'pull-through' technique still restricts the wide use of such an operation. None of the available commercial DMEK injectors could be used for tri-folded DMEK (endothelium-inward) orientation, as it requires the graft to be intently secured within the injector. This report presents a retrospective eye bank validation study of an asymmetrical injector designed to orientally implant a tri-folded DMEK graft without needing a pull-through technique. METHODS: The injector is made from transparent plastic, allowing microscopic tissue validation directly before injection. The device is asymmetrical, so the orientation of the graft can be controlled and validated according to the best eye bank practice, which is critical for successful tri-folded DMEK graft clinical application. Four different designs of the internal compartment of the injectors were evaluated with DMEK tissues. Mates from two pairs were tested on each device type, totaling 16 grafts, all loaded with folded, endo-in grafts. The tissue was prepared, loaded into the injector, and ejected to imitate the tissue manipulation in DMEK operation. RESULTS: After graft loading the delivery of the endothelium-in grafts was performed by injection, without the need for a pull-through technique. One graft (6.25%) has double-scrolled (changed its folding) within the injector with a larger (1.5 mm) internal compartment. The loss of valuable cells was between 3-23% (13.98% average). No significant differences in cell loss were observed between injectors with different internal compartment sizes. Higher viability loss (17.3% +/- 5.7) was observed for the grafts with >20 days death to prep-days in comparison with grafts stored with less than two weeks (10.9% +/-2.1). CONCLUSION: The TissueGUARD injector is the only injector that currently allows oriented, tri-folded DMEK injection without the need for a pull-through technique. The average cell loss after loading and ejection was 13.98%, which is comparable/better than the current best practice with the precut-preloaded technique of naturally folded DMEK.

P16-A111†Factors affecting the density of corneal endothelial cell cultures obtained from donor corneas.

PURPOSE: The shortage of donor corneas represents a worldwide problem, and corneal endothelial cell (CEC) therapy might be a promising alternative approach. CEC can be implanted alone, which has shown limited efficacy, or with a scaffold that holds the cells together as a monolayer tissue, thus imitating Descemet membrane endothelial keratoplasty. We believe that endothelial cell density (ECD) >2000 cells/mm2, a cut-off value that eye banks use to provide quality tissues for transplantation to surgeons, should also be adopted as a parameter to define the quality of CECs as a new Advanced Therapy Medicinal Product for clinical applications in patients with endothelial dystrophies. METHODS: We isolated and cultured CECs from one or more corneas of elderly age donors with ECDs higher than or below 2000 cells/mm2. CEC cultures were carried out on coated plates and on hydrogels with a preformed basement membrane (from TissueGUARD, Germany). Immunofluorescence with antibodies against ZO-1 was performed to evaluate the ECDs of the CEC graft obtained. RESULTS: Our results suggest that primary cultures with ECDs>2000 cells/mm2 can be obtained on coated plated only when (1) CECs are isolated from one or more corneas of young donors; (2) CECs are isolated and pooled together from at least 2 elderly age donor corneas (if ECD>2000 cells/mm2) or 3 elderly age donor corneas (if ECD<2000 cells/mm2). Secondary cultures are all characterized by low ECDs. Hydrogels have been shown to be able to lead to increased ECDs after their release. CONCLUSION: Our protocol highlights the difficulties in obtaining cultures with ECDs>2000 cells/mm2. Despite being achievable with corneas from young donors, this becomes challenging when corneas from elderly donors are used, i.e., the overall majority of those collected by eye banks, particularly when corneas from elderly age donors with ECD<2000 cells/mm2 are considered as a source. One alternative would be to isolate CECs from more corneas, but this might raise the issue of antigenic stimulation, which could eventually lead to transplantation failure. Our strategy to overcome these challenges is the use of a preformed basement membrane as a scaffold for CECs. However, this challenging approach should be investigated more before proceeding to clinical application.

Genomic evolution of vesicular stomatitis virus strains with differences in adaptability.

Virus strains with a history of repeated genetic bottlenecks frequently show a diminished ability to adapt compared to strains that do not have such a history. These differences in adaptability suggest differences in either the rate at which beneficial mutations are produced, the effects of beneficial mutations, or both. We tested these possibilities by subjecting four populations (two controls and two mutants with lower adaptabilities) to multiple replicas of a regimen of positive selection and then determining the fitnesses of the progeny through time and the changes in the consensus, full-length sequences of 56 genomes. We observed that at a given number of passages, the overall fitness gains observed for control populations were larger than fitness gains in mutant populations. However, these changes did not correlate with differences in the numbers of mutations accumulated in the two types of genomes. This result is consistent with beneficial mutations having a lower beneficial effect on mutant strains. Despite the overall fitness differences, some replicas of one mutant strain at passage 50 showed fitness increases similar to those observed for the wild type. We hypothesized that these evolved, high-fitness mutants may have a lower robustness than evolved, high-fitness controls. Robustness is the ability of a virus to avoid phenotypic changes in the face of mutation. We confirmed our hypothesis in mutation-accumulation experiments that showed a normalized fitness loss that was significantly larger in mutant bottlenecked populations than in control populations.

Incongruent fitness landscapes, not tradeoffs, dominate the adaptation of vesicular stomatitis virus to novel host types.

Host radiation refers to the ability of parasites to adapt to new environments and expand or change their niches. Adaptation to one specific environment may involve a loss in adaptation to a second environment. Thus, fitness costs may impose limits to niche expansion and constitute the cost of specialization. Several reports have addressed the cost of host radiation in vesicular stomatitis virus (VSV), but in some cases the experimental setup may have resulted in the overestimation of fitness costs. To clarify this issue, experiments were carried out in which a reference strain of VSV was allowed to adapt to HeLa, MDCK and BHK-21 cells, and to a regime of alternation between HeLa and Madin-Darby canine kidney (MDCK) cells. Measurement of viral fitness on each cell type showed that most virus populations behaved as generalists, and increased in fitness in all environments. Tradeoffs, where a fitness increase in one environment led to a fitness decrease in another environment, were rare. These results highlight the importance of using appropriate methods to measure fitness in evolved virus populations, and provide further support to a model of evolutionary dynamics in which costs due to incongruent landscapes provided by different environments are more common than tradeoffs.