Genetic Blueprint Research Articles

Normalization and the Search for Variation in the Human Genome

This essay reflects on the tension between standardization and the search for variation in the human genome. The stabilization of the human chromosome count in the 1920s was based on the consensus that “Whites,” “Negroes,” and “Japanese,” as well as women and men, had the same number of chromosomes. Yet the idea that there might be chromosomal differences between various groups of people was never quite abandoned. When in the mid-1950s the human chromosome number was revised from 48 to 46, the new count was tested in populations around the world. The description of the “normal human karyotype” that was negotiated in the 1960s was driven by the search for a standard against which the genetic variation revealed by the flurry of testing could be measured. And although the human genome project in the 1990s promised to provide the genetic blueprint that all humans shared, it has in fact led to an increased focus on the genetic variation that distinguishes the history, identity, and health outcomes of various human populations. Following concrete examples, this essay investigates the historically contingent quests that have been driving the search for common standards and variation, and the role Pacific and Indigenous populations have played in these endeavors. This essay is part of a special issue entitled Pacific Biologies: How Humans Become Genetic, edited by Warwick Anderson and M. Susan Lindee.

Screening for texturing Leuconostoc and genomics behind polysaccharide production.

ABSTRACTSynthesis of polysaccharides by Leuconostoc can result in improved texture of fermented products. A total of 249 Leuconostoc strains were screened for homo-polysaccharide production and for texturing capabilities in milk. A total of six Ln. mesenteroides strains with superior texturing properties had the genetic blueprint for both homo- (HoPS) and hetero-polysaccharide (HePS) synthesis. Only one strain produced texture in milk without added sucrose, suggesting HePS synthesis via the Wzy dependent pathway. In milk acidification experiments with added sucrose, all six strains depleted the sucrose and released fructose. Thus, they can be used for both texture and possibly also for sweetness enhancement.

Genetically modified mouse models to help fight COVID-19.

The research community is in a race to understand the molecular mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, to repurpose currently available antiviral drugs and to develop new therapies and vaccines against coronavirus disease 2019 (COVID-19). One major challenge in achieving these goals is the paucity of suitable preclinical animal models. Mice constitute ~70% of all the laboratory animal species used in biomedical research. Unfortunately, SARS-CoV-2 infects mice only if they have been genetically modified to express human ACE2. The inherent resistance of wild-type mice to SARS-CoV-2, combined with a wealth of genetic tools that are available only for modifying mice, offers a unique opportunity to create a versatile set of genetically engineered mouse models useful for COVID-19 research. We propose three broad categories of these models and more than two dozen designs that may be useful for SARS-CoV-2 research and for fighting COVID-19.

Importance of nutrigenomics and nutrigenetics in food Science

Nutrigenomics and nutrigenetics are bidirectional terms that are interrelated as two sides of a coin. Nutritional genomics is the latest scientific discipline that uses modern genomics technology to study the relationship between genes, nutrition, and health. It explores the effects of nutrients on the entire genetic makeup (genome), proteome, and metabolome. Simply, nutrigenomic defines how the diet acts on genes and changes gene expression which is commonly prominent in cancer like non-communicable diseases. Nutrigenetics explains how the genes affect the diet which is generally notable in illnesses like phenylketonuria and lactose intolerance. Nutritional genetics combines the study of nutrition and genetics to discover the different ways people respond to food based on their genetic makeup. Even though humans are similar in genetics, we all have slight differences in our genetic blueprints due to single nucleotide polymorphisms (SNPs) that make us unique from each other. These tiny variations determine both the effect nutrients have on our bodies and how we metabolize the food that we eat. Personalized nutrition connects this two-way relationship between nutrients and genes. This mini-review discusses the applications, advantages, and disadvantages, Sri Lankan context, and future trends of these emerging technologies. Furthermore, this review emphasizes how the consumed nutrients can affect our gene expressions as well as how our genes can influence response to these nutrients. This emerging field could be improved to enhance personalized diet consumption trends depending on individual genetic makeup and to develop innovative functional foods based on genetic patterns. Finally, the production of biomarkers could be applied in the future to predict early disease recognition and to mitigate disease risk.

Housing microbial symbionts: evolutionary origins and diversification of symbiotic organs in animals.

In many animal hosts, microbial symbionts are housed within specialized structures known as symbiotic organs, but the evolutionary origins of these structures have rarely been investigated. Here, I adopt an evolutionary developmental (evo-devo) approach, specifically to apply knowledge of the development of symbiotic organs to gain insights into their evolutionary origins and diversification. In particular, host genetic changes associated with evolution of symbiotic organs can be inferred from studies to identify the host genes that orchestrate the development of symbiotic organs, recognizing that microbial products may also play a key role in triggering the developmental programme in some associations. These studies may also reveal whether higher animal taxonomic groups (order, class, phylum, etc.) possess a common genetic regulatory network for symbiosis that is latent in taxa lacking symbiotic organs, and activated at the origination of symbiosis in different host lineages. In this way, apparent instances of convergent evolution of symbiotic organs may be homologous in terms of a common genetic blueprint for symbiosis. Advances in genetic technologies, including reverse genetic tools and genome editing, will facilitate the application of evo-devo approaches to investigate the evolution of symbiotic organs in animals. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Forensic Genetic Analysis of 11 Rapidly Mutating Y-STRs (RM-YSTRs) in Sindhi Population of Pakistan

Forensic Science is robustly associated with DNA that has a complex genetic blue print information intended to resolve the queries related to civil cases, judiciary, investigatory purposes and crime scenes. The study was carried out to contribute the data set of RMYSTRs (Rapidly Mutating YSTRs) globally. The study focused on the significance of using RM-YSTRs as compared to the set of 9-17 YSTRs (mutation rate 1 × 10-2), as the later one is not being able to attain a high magnitude of male relatives differentiation. In the present work, 100 Sindh DNA male samples were collected, by genotyping the amplified samples, using RM-YSTRs, having a mutation rate of 1 × 10-3. Forensic efficiency parameters such as, PIC (Polymorphism Information Content) was observed in the range of 0.7745 at locus DYS576 to 0.9313 at locus DYF399S1b, maximum gene diversity (0.9350) was found at locus DYF399S1b, minimum gene diversity (0.7978) was seen at locus DYS576. Highest value of allele frequency was found as 0.350 with the allele no 18 at locus DYS576. The results had shown that haplotype frequency and haplotype diversity value were 0.010 and 0.99297, as no haplotype was repeated, which highlighted on the fact that nearly complete male individualization can be obtainedusing RM YSTRs, thus helpful in avoiding the adventitious matches or exclusion or inclusion of male relatives in forensics cases work. It might be an initiative towards establishing RM YSTR database of Pakistani Sindhi population.

Role of Noise-Induced Cellular Variability in Saccharomyces cerevisiae During Metabolic Adaptation: Causes, Consequences and Ramifications

The concept of genetic determinism as illustrated by the metaphors such as ‘genetic blueprint’, or ‘genetic program’ had its beginning immediately after the rediscovery of growth lag observed when bacteria were exposed to a mixture of glucose and galactose or lactose. This concept got reinforced with the discovery of the mechanisms of how lactose activates the transcription of lac operon of E. coli. According to this doctrine, genetically identical cells exposed to the same environment respond in equal measure. However, studies carried out in the past two decades in organisms ranging from prokaryotes to eukaryotes, have clearly established that genetically identical cells need not necessarily respond in an identical fashion when exposed to a given environment. It has now become amply clear that organisms can stochastically switch from one physiological state to the other, thereby resulting in a phenotypically heterogeneous population. Such exhibition of heterogeneity by a population has been, in several contexts, shown to be beneficial in a temporally changing environment. In this review, we have discussed how individual cells of a genetically identical population of Saccharomyces cerevisiae remain fit by exploiting this fascinating phenomenon of stochastic switching from one metabolic state to the other when exposed to glucose and galactose, as a source of carbon and energy. We suggest that this inherent stochastic switching seems to have been exploited for an adaptive response in a fluctuating environment.

The role of inflammation and genetics in periodontal disease.

Periodontitis is a complex disease: (a) various causative factors play a role simultaneously and interact with each other; and (b) the disease is episodic in nature, and bursts of disease activity can be recognized, ie, the disease develops and cycles in a nonlinear fashion. We recognize that various causative factors determine the immune blueprint and, consequently, the immune fitness of a subject. Normally, the host lives in a state of homeostasis or symbiosis with the oral microbiome; however, disturbances in homeostatic balance can occur, because of an aberrant host response (inherited and/or acquired during life). This imbalance results from hyper‐ or hyporesponsiveness and/or lack of sufficient resolution of inflammation, which in turn is responsible for much of the disease destruction seen in periodontitis. The control of this destruction by anti‐inflammatory processes and proresolution processes limits the destruction to the tissues surrounding the teeth. The local inflammatory processes can also become systemic, which in turn affect organs such as the heart. Gingival inflammation also elicits changes in the ecology of the subgingival environment providing optimal conditions for the outgrowth of gram‐negative, anaerobic species, which become pathobionts and can propagate periodontal inflammation and can further negatively impact immune fitness. The factors that determine immune fitness are often the same factors that determine the response to the resident biofilm, and are clustered as follows: (a) genetic and epigenetic factors; (b) lifestyle factors, such as smoking, diet, and psychosocial conditions; (c) comorbidities, such as diabetes; and (d) local and dental factors, as well as randomly determined factors (stochasticity). Of critical importance are the pathobionts in a dysbiotic biofilm that drive the viscious cycle. Focusing on genetic factors, currently variants in at least 65 genes have been suggested as being associated with periodontitis based on genome‐wide association studies and candidate gene case control studies. These studies have found pleiotropy between periodontitis and cardiovascular diseases. Most of these studies point to potential pathways in the pathogenesis of periodontal disease. Also, most contribute to a small portion of the total risk profile of periodontitis, often limited to specific racial and ethnic groups. To date, 4 genetic loci are shared between atherosclerotic cardiovascular diseases and periodontitis, ie, CDKN2B‐AS1(ANRIL), a conserved noncoding element within CAMTA1 upstream of VAMP3, PLG, and a haplotype block at the VAMP 8 locus. The shared genes suggest that periodontitis is not causally related to atherosclerotic diseases, but rather both conditions are sequelae of similar (the same?) aberrant inflammatory pathways. In addition to variations in genomic sequences, epigenetic modifications of DNA can affect the genetic blueprint of the host responses. This emerging field will yield new valuable information about susceptibility to periodontitis and subsequent persisting inflammatory reactions in periodontitis. Further studies are required to verify and expand our knowledge base before final cause and effect conclusions about the role of inflammation and genetic factors in periodontitis can be made.

Asian Zika Virus Isolate Significantly Changes the Transcriptional Profile and Alternative RNA Splicing Events in a Neuroblastoma Cell Line.

The alternative splicing of pre-mRNAs expands a single genetic blueprint to encode multiple, functionally diverse protein isoforms. Viruses have previously been shown to interact with, depend on, and alter host splicing machinery. The consequences, however, incited by viral infection on the global alternative slicing (AS) landscape are under-appreciated. Here, we investigated the transcriptional and alternative splicing profile of neuronal cells infected with a contemporary Puerto Rican Zika virus (ZIKVPR) isolate, an isolate of the prototypical Ugandan ZIKV (ZIKVMR), and dengue virus 2 (DENV2). Our analyses revealed that ZIKVPR induced significantly more differential changes in expressed genes compared to ZIKVMR or DENV2, despite all three viruses showing equivalent infectivity and viral RNA levels. Consistent with the transcriptional profile, ZIKVPR induced a higher number of alternative splicing events compared to ZIKVMR or DENV2, and gene ontology analyses highlighted alternative splicing changes in genes associated with mRNA splicing. In summary, we show that ZIKV affects cellular RNA homeostasis not only at the transcriptional levels but also through the alternative splicing of cellular transcripts. These findings could provide new molecular insights into the neuropathologies associated with this virus.

Histological changes, Micronuclei induction and Nuclear abnormalities in the peripheral erythrocytes of Clarias gariepinus (Burchell 1822) exposed to water sample from Apodu Reservoir

Pollution of water bodies in most developing countries is of grave concern since toxic pollutants interact with the genetic blueprints of aquatic organisms leading to a variety of illnesses. In this study, histological changes in the gills, liver and kidney as well as the genotoxic effect of water sample from Apodu reservoir, were investigated in the peripheral erythrocytes of cultured catfish (Clarias gariepinus) using micronucleus assay. They were exposed to the water sample for a period of 3, 7 and 14 days. The physico-chemical (pH, temperature, transparency) and heavy metals analysed (Ca>Mg>Fe>Zn>Mn>Cu>Ni=Pb=Cd=Cr) were all within the acceptable limit permissible. However, the micronucleus test shows statistically significant (p<0.05) induction of micronuclei and nuclear abnormalities as compared to the control (borehole water). The histopathological analysis shows some mild alterations in the organs such as distortion and infiltration of lymphocytic cells of the gills. We also reported mild distortion of hepatic tissue of the liver as well as abnormal nephron and glomeruli with congestion and vascular dilation of the kidney. These alterations might be due to anthropogenic activities that pollute the water body. Therefore, the influx of agricultural and domestic waste into the Apodu reservoir should be discouraged as part of remediating efforts.

Variation of Glycosylation Patterns Revealed by PGC‐LC‐ and Gated‐TIMS‐EED Tandem Mass Spectrometry

IntroductionGlycans are the most abundant biopolymers found in nature; they play crucial roles in a wide range of biological activities. Glycan biosynthesis does not follow genetic blueprints, and the resultant glycome is often a mixture of glycoforms with varying compositions, branching patterns, linkages, and stereochemical configurations. Subtle changes in the structure of a glycan can have a profound impact on its biological functions. Recently, we have demonstrated the potential of porous graphitic carbon liquid chromatography electronic excitation dissociation tandem mass spectrometry (PGC‐LC EED MS/MS) and gated‐trapped ion mobility spectrometry (TIMS) negative electron transfer dissociation (NETD) MS/MS in characterizing glycan mixtures, including the separation and identification of isomeric structures. Here, by studying N‐glycans released from different batches of glycoproteins, the glycoform variations at the isomer level were observed. The detailed structure of each isomer could be determined by EED MS/MS and their relative quantification may be achieved by high‐resolution LC or ion mobility separation.MethodsThe released N‐linked glycans were reduced and permethylated before analysis to improve the ionization efficiency and to facilitate spectral interpretation. All experiments were carried out on a 12T solariXTM Fourier‐transform ion cyclotron resonance mass spectrometer equipped with a prototype TIMS device (Bruker Daltonics) or a nanoACQUITY UPLC system (Waters).ResultsRibonuclease B (RNase B) is the glycosylated form of RNase A. With the attachment of a glycan at Asn‐34, the glycosylated ribonuclease showed an increased structural flexibility in the RNA binding region and a much higher activity on double‐stranded RNAs. Previously, application of PGC‐EED MS/MS to the analysis of N‐glycans released from RNase B showed the presence of 18 high‐mannose structures. Here, by comparing the PGC‐LC MS/MS results from two batches of RNase B purchased from different manufacturers, we observed variations in the abundance of each glycoform. Additionally, we have implemented EED MS/MS with gated‐TIMS separation, and showed its ability to resolve and identify isomeric structures. Using gated‐TIMS EED MS/MS, we were able to determine the batch‐to‐batch variation of chicken ovalbumin N‐glycans along with the conformational information.ConclusionsThe PGC‐ and gated‐TIMS‐ EED MS/MS approaches showed advanced performance in separation, characterization, and relative quantification of isomeric glycans, as well as in applications to determine the batch‐to‐batch glycoform variations of glycoproteins. These approaches hold great potential for comprehensive and confident glycan characterization.Support or Funding InformationThis research is supported by the NIH grants P41 GM104603, 703 R21 GM122635, U01CA221234, R01 GM132675, and S10 704 RR025082.

Ribosome-guided piRNA production.

The ribosome decodes messenger RNAs and constructs proteins based on the genetic blueprint. Ribosomes also associate with non-coding RNAs, such as PIWI-interacting RNA (piRNA) precursors, during the meiotic pachytene stage. Intriguingly, the ribosome mediates pachytene piRNA biogenesis by guiding endonucleolytic cleavage of piRNA precursors.

Genetic test for the prescription of diets in support of physical activity.

Owing to the fields of nutrigenetics and nutrigenomics today we can think of devising approaches to optimize health, delay onset of diseases and reduce its severity according to our genetic blue print. However this requires a deep understanding of nutritional impact on expression of genes that may result in a specific phenotype. The extensive research and observational studies during last two decades reporting interactions between genes, diet and physical activity suggest a cross talk between various genetic and environmental factors and lifestyle interventions. Although considerable efforts have been made in unraveling the mechanisms of gene-diet interactions the scientific evidences behind developing commercial genetic tests for providing personalized nutrition recommendations are still scarce. In this scenario the current mini-review aims to provide useful insights into salient feature of nutrition based genetic research and its commercial application and the ethical issue and concerns related to its outcome. (www.actabiomedica.it)

Development of the brainstem respiratory circuit.

The respiratory circuit is comprised of over a dozen functionally and anatomically segregated brainstem nuclei that work together to control respiratory rhythms. These respiratory rhythms emerge prenatally but only acquire vital importance at birth, which is the first time the respiratory circuit faces the sole responsibility for O2 /CO2 homeostasis. Hence, the respiratory circuit has little room for trial-and-error-dependent fine tuning and relies on a detailed genetic blueprint for development. This blueprint is provided by transcription factors that have specific spatiotemporal expression patterns along the rostral-caudal or dorsal-ventral axis of the developing brainstem, in proliferating precursor cells and postmitotic neurons. Studying these transcription factors in mice has provided key insights into the functional segregation of respiratory control and the vital importance of specific respiratory nuclei. Many studies converge on just two respiratory nuclei that each have rhythmogenic properties during the prenatal period: the preBötzinger complex (preBötC) and retrotrapezoid nucleus/parafacial nucleus (RTN/pF). Here, we discuss the transcriptional regulation that guides the development of these nuclei. We also summarize evidence showing that normal preBötC development is necessary for neonatal survival, and that neither the preBötC nor the RTN/pF alone is sufficient to sustain normal postnatal respiratory rhythms. Last, we highlight several studies that use intersectional genetics to assess the necessity of transcription factors only in subregions of their expression domain. These studies independently demonstrate that lack of RTN/pF neurons weakens the respiratory circuit, yet these neurons are not necessary for neonatal survival because developmentally related populations can compensate for abnormal RTN/pF function at birth. This article is categorized under: Nervous System Development > Vertebrates: Regional Development.

DNA‐Guided Assembly of Molecules, Materials, and Cells

DNA is the genetic blueprint for most known living organisms on Earth, but it is not merely the secret of life. Because of its programmability derived from Watson–Crick base‐pairing, DNA exhibits unparalleled versatility in constructing designer molecular structures by self‐assembly—a field called DNA nanotechnology. After decades of active pursuit, DNA has demonstrated unprecedented capability in designing highly prescribed and sophisticated artificial nanostructures, which could be either static, with well‐controlled physicochemical properties, or dynamic, with the ability to change in response to external stimuli. Researchers have devoted considerable efforts to exploring the utility of DNA nanotechnology in a variety of fields, with the majority related to harvesting their assembling power, since they are highly capable of guiding the assembly of other entities—molecules, materials, living cells, and so on—to fabricate artificial assemblies with emergent properties and functions. Herein, a brief introduction is given to self‐assembly methods in DNA nanotechnology, including DNA tiles, modular DNA bricks, and DNA origami. Four sections are then dedicated to covering the utilization of DNA nanotechnology for guided assembly of organic materials, inorganic materials, biological materials, and living cells. Lastly, DNA‐enabled molecular intelligent systems that are able to compute and execute algorithms are discussed.

Aptamers as Modular Components of Therapeutic Nucleic Acid Nanotechnology.

Nucleic acids play a central role in all domains of life, either as genetic blueprints or as regulators of various biochemical pathways. The chemical makeup of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), generally represented by a sequence of four monomers, also provides precise instructions for folding and higher-order assembly of these biopolymers that, in turn, dictate biological functions. The sequence-based specific 3D structures of nucleic acids led to the development of the directed evolution of oligonucleotides, SELEX (systematic evolution of ligands by exponential enrichment), against a chosen target molecule. Among the variety of functions, selected oligonucleotides named aptamers also allow targeting of cell-specific receptors with antibody-like precision and can deliver functional RNAs without a transfection agent. The advancements in the field of customizable nucleic acid nanoparticles (NANPs) opened avenues for the design of nanoassemblies utilizing aptamers for triggering or blocking cell signaling pathways or using aptamer-receptor combinations to activate therapeutic functionalities. A recent selection of fluorescent aptamers enables real-time tracking of NANP formation and interactions. The aptamers are anticipated to contribute to the future development of technologies, enabling an efficient assembly of functional NANPs in mammalian cells or in vivo. These research topics are of top importance for the field of therapeutic nucleic acid nanotechnology with the promises to scale up mass production of NANPs suitable for biomedical applications, to control the intracellular organization of biological materials to enhance the efficiency of biochemical pathways, and to enhance the therapeutic potential of NANP-based therapeutics while minimizing undesired side effects and toxicities.

Himalayan Saccharomyces eubayanus Genome Sequences Reveal Genetic Markers Explaining Heterotic Maltotriose Consumption by Saccharomyces pastorianus Hybrids.

Saccharomyces pastorianus strains are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus that have been domesticated for centuries in lager beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origins of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus × S. cerevisiae hybrids. To address this question, we generated a nearly complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains has been proposed to be the S. eubayanus subgenome origin of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of an S. eubayanusAGT1 (SeAGT1) α-oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus Although Himalayan S. eubayanus strains cannot grow on maltose and maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose transporter genes complemented the corresponding null mutants of S. cerevisiae Expression, in Himalayan S. eubayanus of a functional S. cerevisiae maltose metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing an S. cerevisiae × S. eubayanus laboratory hybrid with a complement of maltose metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume maltotriose in brewer's wort demonstrated regulatory cross talk between subgenomes and thereby validated this hypothesis. These results support experimentally the new postulated hypothesis on the evolutionary origin of an essential phenotype of lager brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids.IMPORTANCES. pastorianus, an S. cerevisiae × S. eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide. It emerged by spontaneous hybridization and colonized early lager brewing processes. Despite accumulation and analysis of genome sequencing data of S. pastorianus parental genomes, the genetic blueprint of industrially relevant phenotypes remains unresolved. Assimilation of maltotriose, an abundant sugar in wort, has been postulated to be inherited from the S. cerevisiae parent. Here, we demonstrate that although Asian S. eubayanus isolates harbor a functional maltotriose transporter SeAGT1 gene, they are unable to grow on α-oligoglucosides, but expression of S. cerevisiae regulator MAL13 (ScMAL13) was sufficient to restore growth on trisaccharides. We hypothesized that the S. pastorianus maltotriose phenotype results from regulatory interaction between S. cerevisiae maltose transcription activator and the promoter of SeAGT1 We experimentally confirmed the heterotic nature of the phenotype, and thus these results provide experimental evidence of the evolutionary origin of an essential phenotype of lager brewing strains.

Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

Pharmacogenomics in clinical care

Health care designed specifically for a person based on their genetic makeup ("personalized" or "precision" medicine) is expanding rapidly, especially in the area of drug selection. Pharmacogenomic (PGx) testing, when drugs and doses are selected based on an individual's genetic profile, is increasingly being used to guide the selection of drugs or therapies to optimize outcomes and minimize side effects. Based on an individual's genetic blueprint, health care providers now have important information about how a drug is likely to behave in that individual's body. Pharmacogenomic information on drug labels is now available for nearly 250 drugs. Health care organizations are also increasingly making this information available to customers to reduce emergency department visits, improve outcomes (selecting the right chemotherapy doses), and reduce cost. This study reviews some of the challenges and benefits on using PGx testing to improve clinical outcomes.

Genes and genetics in hyperhomocysteinemia and the "1-carbon metabolism": implications for retinal structure and eye functions.

Homocysteine (Hcy), a sulfur-containing nonproteinogenic amino acid, is generated as a metabolic intermediate. Hcy constitutes an important part of the "1-carbon metabolism" during methionine turnover. Elevated levels of Hcy known as hyperhomocysteinemia (HHcy) results from vitamin B deficiency, lack of exercise, smoking, excessive alcohol intake, high-fat and methionine-rich diet, and the underlying genetic defects. These factors directly affect the "1-carbon metabolism (methionine-Hcy-folate)" of a given cell. In fact, the Hcy levels are determined primarily by dietary intake, vitamin status, and the genetic blueprint of the susceptible individual. Although Hcy performs an important role in cellular functions, genetic alterations in any of the key enzymes responsible for the "1-carbon metabolism" could potentially upset the metabolic cycle, thus causing HHcy environment in susceptible people. As such, HHcy relates to several clinical conditions like atherosclerosis, myocardial infarction, stroke, cognitive impairment, dementia, Parkinson's disease, multiple sclerosis, epilepsy, and ocular disorders, among others. This article summarizes the findings from our laboratory and public database regarding genetics of HHcy and its effects on ocular disorders, their respective management during dysregulation of the 1-carbon metabolism.