While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types. ll

There is now compelling evidence that microbially mediated reactions impart a significant effect upon the dynamics, composition, and abundance of nutrients in glacial melt water. Consequently, we must now consider ice masses as ecosystem habitats in their own right and address their diversity, functional potential, and activity as part of alpine and polar environments. Although such research is already underway, its fragmentary nature provides little basis for developing modern concepts of glacier ecology. This paper therefore provides a much-needed framework for development by reviewing the physical, biogeochemical, and microbiological characteristics of microbial habitats that have been identified within glaciers and ice sheets. Two key glacial ecosystems emerge, one inhabiting the glacier surface (the supraglacial ecosystem) and one at the ice-bed interface (the subglacial ecosystem). The supraglacial ecosystem is characterized by a diverse consortium of microbes (usually bacteria, algae, phytoflagellates, fungi, viruses and occasional rotifers, tardigrades, and diatoms) within the snowpack, supraglacial streams, and melt pools (cryoconite holes). The subglacial system is dominated by aerobic/anaerobic bacteria and most probably viruses in basal ice/till mixtures and subglacial lakes. A third, so-called englacial ecosystem is also described, but it is demonstrated that conditions within glacier ice are sufficient to make metabolic activity and its impact upon nutrient dynamics negligible at the glacier scale.Consideration of the surface and internal heat balances of the glacier show that all glacial ecosystems are sensitive to climate change, although at different timescales. Thus, while rapid, melt-driven habitat changes lead to melt-out, resuscitation, and redistribution of microorganisms in many supraglacial ecosystems, much slower climatic and glacial mass-balance processes effect such changes in the subglacial ecosystem. Paradoxically, it is shown that these forces have brought about net refreezing and the onset of cryostasis in the subglacial ecosystems of many Arctic glaciers subject to thinning in recent decades.

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposonrelated genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures. STEM CELLS 2007;25:371-379

Background Recessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7−/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans. Methods Between October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography. Results One patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal–epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies. Conclusions Increased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

Highlights d Engineered virus-like particles (eVLPs) overcome three bottlenecks to protein delivery d DNA-free eVLPs efficiently deliver gene editing proteins with minimal off-target editing d Base editor eVLPs reduced serum Pcsk9 levels 78% following 63% liver editing in mice d Base editor eVLPs improved visual function in a mouse model of genetic blindness