Rewriting Human History and Empowering Indigenous Communities with Genome Editing Tools.

Appropriate empirical-based evidence and detailed theoretical considerations should be used for evolutionary explanations of phenotypic variation observed in the field of human population genetics (especially Indigenous populations). Investigators within the population genetics community frequently overlook the importance of these criteria when associating observed phenotypic variation with evolutionary explanations. A functional investigation of population-specific variation using cutting-edge genome editing tools has the potential to empower the population genetics community by holding "just-so" evolutionary explanations accountable. Here, we detail currently available precision genome editing tools and methods, with a particular emphasis on base editing, that can be applied to functionally investigate population-specific point mutations. We use the recent identification of thrifty mutations in the CREBRF gene as an example of the current dire need for an alliance between the fields of population genetics and genome editing

Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors.

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer

Selective Cytotoxicity of Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anticancer agents

Targeted Chemotherapy with Metal Complexes

Classical chemotherapeutics, such as cisplatin and its analogues, have been highly successful in the clinic, yet improvements can certainly be made, given the significant side effects associated with the killing of healthy cells. Recent advances in the field of chemotherapy include the development of targeted anticancer agents, compounds that are directed towards a specific biomarker of cancer, with the hopes that such targeted therapies might have reduced side effects given their greater selectivity. Here we discuss several transition metal complexes that are tailored towards various biomolecules associated with cancer. Most notably, the success of rhodium metalloinsertors, which specifically bind to nucleic acid base mismatches in DNA, highlight the enormous potential of this exciting new strategy

Chemical Quality of Base Flow in 18 Selected Streams in the Upper Susquehanna River Basin, New York

The base-flow and runoff components of total streamflow at four selected sites in the upper Susquehanna River Basin in New York were calculated through hydrograph-separation techniques from long-term (1941-93) discharge records. Base flow was found to constitute more than 60 percent of the total annual flow of each stream. Base-flow samples were then collected at 18 stream sites several times during 2001 to define the chemical quality of base flow. The concentrations of selected common ions, nutrients, and pesticides were plotted in relation to the amount of agricultural land and carbonate bedrock in the drainage basin upstream of each site. Sites were selected at locations distant from and unaffected by development and urban areas. Twelve of the sites were again sampled in November 2001 for pesticide analysis. The predominant cations detected in the samples were calcium, magnesium, and sodium; the major anions were chloride, sulfate, and bicarbonate. The predominant nutrient was nitrate. Higher nitrate concentrations in the winter samples than in the summer samples are attributed to the seasonal decrease in plant growth and microbial activity in the streams during the winter, which allows nitrate to persist in the stream water. Lower nitrate concentrations in the summer samples probably result from nitrogen uptake by vegetation and microbial activity in the streams. Base-flow samples from the agricultural, carbonate-rich northern part of the study area had higher concentrations of most inorganic chemical constituents than those from the forested, noncarbonate (shale, siltstone, and sandstone) central and southern parts. The highest nitrate concentrations were in samples from subbasins dominated by agricultural land, and the lowest were in subbasins dominated by forest. The concentrations in samples from subbasins with forested as well as agricultural land were intermediate. Six pesticides were detected in samples from 10 of the 12 sites. All were herbicides. The highest concentrations of pesticides, and the most frequent pesticide detections, were in samples from agricultural subbasins and large main-stem subbasins with mixed land use and mixed bedrock geology. A correlation was indicated between land use and concentrations of atrazine and deethylatrazine. The concentrations of all six compounds were at least an order of magnitude lower than New York State and Federal water-quality standards. Ground water from four production wells in the villages of Afton, Sidney, Unadilla, and Otego was analyzed for chlorofluorocarbons (CFCs) to indicate the approximate age of the water in these wells and the potential for induced infiltration of river water. The water at two of these wells is probably between 26 and 50 years old; the ages of water at the other two wells could not be reliably estimated because of CFC contamination from a nonatmospheric source. The two wells for which CFC analysis gave reliable results (Afton and Otego) probably do not induce infiltration of river water into the aquifer

Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA