path: root/gnqa/paper1_eval/src/data/datasets/human/dataset_domainexpert_general_2.json

{
  "question": [
    "Once a sperm combines with an egg, what determines how traits are passed onto the resulting lifeform?",
    "Why is genetic tracing matrilineal rather than patrilineal?",
    "Explain the process of DNA replication and how it ensures accurate copying of genetic information during cell division.",
    "What are the potential benefits and risks associated with gene editing technologies like CRISPR-Cas9?",
    "How does one tell the difference between X and Y DNA, with repsect to DNA tracing and determining QTLs?"
  ],
  "answer": [
    "The traits are determined by the combination of genes from both the sperm and the egg. This process involves meiosis, where each gamete (sperm and egg) contributes one chromosome to each pair, resulting in a zygote with a full complement of 23 chromosome pairs. The process of recombination or crossing over, where similar DNA sequences from the paired chromosomes swap genetic material, also plays a crucial role in determining the traits of the offspring. This results in a shuffling of genetic material and contributes to the genetic variation seen among offspring.",
    "Genetic tracing is matrilineal due to the inheritance of mitochondrial DNA (mtDNA), which is passed from mother to all her children without any admixture from the father. This allows for a clear lineage tracing through the maternal line. In contrast, Y-DNA is passed from father to son, allowing for patrilineal tracing, but it does not provide information about the genetic contributions of other ancestors in a family tree.",
    "DNA replication is a process where the DNA molecule creates two identical copies of itself. This process begins with the separation of the two strands of the mother cell DNA. New nucleotides are then assembled to form two double helices identical to the original one. This is facilitated by the base pairing rules where adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). This ensures that each daughter cell receives an exact copy of the DNA. The replication process is crucial during cell division as it allows for the accurate transmission of genetic information from one generation of cells to the next.",
    "The potential benefits of gene editing technologies like CRISPR-Cas9 include the ability to modify genes for the treatment of diseases, improvement of crop species, and the development of personalized drug or cell therapies. It can also be used for functional screening in the development of therapies and for the study of molecular causes of ageing. However, there are risks associated with these technologies. These include off-target effects, which can lead to unwanted mutations, and the potential for wide-ranging deletions or recombination events. There's also a risk of triggering a P53 response leading to apoptosis in cycling cells, and the potential for subjects to generate antibodies to Cas9, which could limit gene therapies. Furthermore, the long-term safety of CRISPR genome editing in humans is yet to be determined.",
    "The text does not provide specific information on how to differentiate between X and Y DNA in the context of DNA tracing and determining QTLs."
  ],
  "contexts": [
    [
      "Selection could occur at multiple levels, from germ cell generation and propagation to fertilization and early embryonic growth.Chromosomal abnormalities, including aneuploidy, were found in 10-20% of spermatozoa and oocytes (20) and in the cleaved embryo, with a 21% rate of abnormalities in preimplantation embryos (21).These findings led to a model for natural selection against chromosome abnormalities (21).Selection extends to the end of gestation: Only approximately 30% of all conceptions result in a live birth, with more than half of aborted fetuses containing chromosomal abnormalities (22), a number likely to be an underestimate because of technological limitations in measuring all possible mutations.But even in the very small fraction of germ cell duos that survive this withering genome attack and result in a live birth, a number of severe de novo mutations will still be found (23).The data on gross chromosomal alterations suggest that overall, mutation frequency early in life is very high.The functional consequence, however, is limited because of selection.Somewhat surprisingly, this picture points toward an initial decline in genomic alterations, allowing the adult individual to acquire a somatic genome optimally equipped to provide function.",
      "The phenotype ofthe F1 hybrids is compared to those of the parental inbred strains to revealdominance or semi-dominance relationships between the alleles that aect thephenotype. Phenotypic dierences between reciprocal F1 hybrids indicate thatone or more of the following factors may aect the trait: (1) sex linkage (X- or Ylinked traits), (2) genomic imprinting of QTLs that aect the phenotype, (3)prenatal maternal eects (eects of intrauterine environment), and/or (4)postnatal maternal or paternal eects (eects of maternal and/or paternalparenting behaviour on ospring).",
      "Sex brings harmful alleles together into thesame genetic background, allowing selection to more efficiently purge them fromthe population and potentially producing some offspring that are fitter than eitherparent. However, the benefit of recombining deleterious mutations may depend on thenature of the epistatic interactions between them. The mutational deterministic hypothesis(Kondrashov 1988) depends partly on this epistasis.In most plants and animals, sexis a necessary component of reproduction, and the question for evolutionary biologistsis why reproductive mechanisms have evolved that way. In one of the experimentsdescribed next, evolutionary geneticists have nevertheless devised a way to compareevolution with and without recombination in the obligately sexual fruit fly.This disparity in investment is the basis for the twofold cost: asexualfemales hypothetically could transmit twice as many alleles at the same cost. In most plants and animals, mates tend to be unrelated, leading to outcrossing. Butsex usually also involves the basic process of physical recombination: the breakage andreunion of two different DNA or RNA molecules. Of these two processes, recombinationis clearly the more widespread feature of sexual reproduction. A variety of reproductivesystems, such as selfing and automixis, involve recombination but not outcrossing. Incontrast, relatively few reproductive systems have outcrossing without recombination.",
      "Crossing over-The swapping of genetic material that occurs in the germline.During the formation of egg and sperm cells, also known as meiosis, paired chromosomes from each parent align so that similar DNA sequences from the paired chromosomes cross over one another.Crossing over results in a shuffling of genetic material and is an important cause of the genetic variation seen among offspring.This process is also known as meiotic recombination.The reason for the rarity of these mutations is natural selection: If the mutations result in disorders that decrease health and reproductive fitness, they will eventually be eliminated from a population.In exceptional cases, mutations may cause both beneficial and detrimental consequences, resulting in opposing forces of positive selection and negative selection that may cause the mutations to be preserved at nonrare frequencies in a population.For example, the HbS mutation in the HBB gene (which produces the  subunit of hemoglobin) causes sickle cell disease when present in both alleles, a detrimental consequence, but protects against malaria when present in 1 allele, a beneficial consequence, ensuring that the mutation persists in populations in areas of the world where malaria is endemic.Genes are passed from parents to offspring via the process of meiosis by which gametes, the egg cells in the mother and the sperm cells in the father, are generated.Ordinarily, each cell has 23 pairs of chromosomes; the gametes have 23 unpaired chromosomes.In meiosis, the 23 pairs are split so that each gamete receives 1 chromosome from each pair (Figures 8 and 9).Two gametes (egg and sperm) ultimately join into a single cell, the zygote, which has the full complement of 23 chromosome pairs restored.If all goes well, the zygote gives rise to a live offspring.Recombination (meiotic recombination)-The swapping of genetic material that occurs in the germline.During the formation of egg and sperm cells, also known as meiosis, paired chromosomes from each parent align so that similar DNA sequences from the paired chromosomes recombine with one another.Recombination results in a shuffling of genetic material and is an important cause of the genetic variation seen among offspring.Also known as crossing over.",
      "In the generation of gametes, crossing over regularly occurs, and genetic information is swapped between members of a chromosome pair.That doesn't matter within inbred animals, because the swapped parts are identical.In an F 1 animal, however, the chromosomes of a particular pair are genetically different, one each having come from each parent.Each gamete produced will be unique, as will be each F 2 zygote formed by uniting of the gametes from two F 1 parents.An F 2 group thus provides for expression of some genetic variability.This variability is limited to the allelic differences existing between the parent strains of the F 1 s, so that another F 2 , derived from different inbred strains, will express different genetic differences.",
      "Sex brings harmful alleles together into thesame genetic background, allowing selection to more efficiently purge them fromthe population and potentially producing some offspring that are fitter than eitherparent. However, the benefit of recombining deleterious mutations may depend on thenature of the epistatic interactions between them. The mutational deterministic hypothesis(Kondrashov 1988) depends partly on this epistasis.In most plants and animals, sexis a necessary component of reproduction, and the question for evolutionary biologistsis why reproductive mechanisms have evolved that way. In one of the experimentsdescribed next, evolutionary geneticists have nevertheless devised a way to compareevolution with and without recombination in the obligately sexual fruit fly.This disparity in investment is the basis for the twofold cost: asexualfemales hypothetically could transmit twice as many alleles at the same cost. In most plants and animals, mates tend to be unrelated, leading to outcrossing. Butsex usually also involves the basic process of physical recombination: the breakage andreunion of two different DNA or RNA molecules. Of these two processes, recombinationis clearly the more widespread feature of sexual reproduction. A variety of reproductivesystems, such as selfing and automixis, involve recombination but not outcrossing. Incontrast, relatively few reproductive systems have outcrossing without recombination.",
      "Aberrant recombination patterns on chromosomes that have missegregated have also been identified as an important factor, in both male and female gametes (Table I).This is because recombination together with cohesion of sister chromatids establish the unique 'bivalent' chromosome structure where homologous partner chromosomes are tethered together, a configuration that is critical for their accurate segregation in meiosis I (Fig. 2A).The remarkable feature is that recombination occurs in foetal oocytes whereas chromosome segregation takes place decades later (Fig. 2A).Since mammalian oocytes are arrested at the G2/M transition (or dictyate stage), this raises the intriguing question of how the bivalent is maintained until the meiotic divisions.",
      "Traditionally, it has been agreed that thenal sex of an individual (phenotypic sex)depends on two sequential processes: the sexdetermination system of the species and thegonad differentiation process (Valenzuela,2008). However, recently, these two seeminglydistinct processes are viewed as part of a general process leading to gonad formation andsex ratios (Sarre et al. , 2004; Quinn et al. , 2011;Uller and Helantera, 2011).However, we expect thatonly at this level, the most signicant contributions brought by integrating epigenetics will bemade. Concluding Remarks and FutureProspectsFish sex ratios are the result of a complex interaction between genetic, biochemical, and environmental interactions. The ultimate resultof these interactions at the individual level isgender: male or female. However, at the population level, the combination of sex determination and differentiation sets the sex ratio. Inturn, sex ratios dene the reproductive capacityof populations and, if sex growth dimorphismexists, also the growth characteristics, something very important in an aquaculture context.",
      "Obehav is, in turn, influenced by offspring genesand environment (Ogene and Oenvir respectively). Hence, indirect genetic effects (blue arrows)and direct genetic effects (red arrow) are important influencers of behaviour. B) Parentoffspring conflict theory predicts that parental resource investment and offspring solicitationbehaviours are influenced by the fitness benefit to a focal individual (O), cost to a socialpartner such as a sibling (S1 and S2) or parent (P), and by their coefficient of relatedness(black arrows). 42Figure 2: Genomic imprinting can result in divergent phenotypes from the samegenotype. A) A paternally imprinted gene, i.e. maternally expressed.",
      "Because of the small contribution, through the sperm, ofthe paternal transcriptome to the fertilized zygote, and because of the stronger maternal contributionto child rearing in most model organisms, parental effects are typically thought of as synonymous withmaternal effects, although true paternal effects are known to exist (Rando, 2012). Maternal effects have been shown to be important during embryonic development, leading todifferences in the birth weight of mice depending on the genotype of the mother (Cowley et al. ,1989; Wolf et al. , 2011).Therefore, the resulting phenotypic patterns lag a generationbehind the genetic transmission of the causal variants. The most well-studied parental genetic effectsare caused by deposition of maternal transcripts into the egg prior to fertilization, resulting indifferences in early embryonic development depending on the genotype of the mother. Certain geneshave also been shown to respond to maternal influence after birth through genetically definedmaternal behaviors (Weaver et al. , 2004).",
      "The phenotype ofthe F1 hybrids is compared to those of the parental inbred strains to revealdominance or semi-dominance relationships between the alleles that aect thephenotype. Phenotypic dierences between reciprocal F1 hybrids indicate thatone or more of the following factors may aect the trait: (1) sex linkage (X- or Ylinked traits), (2) genomic imprinting of QTLs that aect the phenotype, (3)prenatal maternal eects (eects of intrauterine environment), and/or (4)postnatal maternal or paternal eects (eects of maternal and/or paternalparenting behaviour on ospring).",
      "It was believed by many that for each trait variant we should expect to find acorresponding genetic change, or gene for that trait. Through historical happenstance therelationship between genes and traits was set up and treated as if it were one-to-one. But theproduction of a trait involves not only genes, but also their interactions with each other and theenvironment, and chance."
    ],
    [
      "distinguishing prenatalfrom postnatal maternal effects, see below). Maternal effects canaccount for a large proportion of phenotypic variance, especiallyduring early life, and for some traits explain more variation thandirect genetic effects [33, 97, 99, 100, 102115]. However, maternal and offspring genotype are correlated (i.e. half their genes areshared), and in inbred lines they are fully confounded, thus separating the effects of their respective genotypes is difficult. To removethis confounding effect cross-fostering has been used, both in thelaboratory and in the field [119, 131].",
      "Using genetic markers, the pattern of inheritance can be tracked throughfamilies. For example, by analyzing a marker linked to the eye color genein several generations, it is possible to determine from which grandparents achild has inherited its eye color alleles. More importantly, nding a markerlinked to a disease can lead to location of the faulty gene causing the disease. Finding the gene is very valuable in the search for the cure. The distance between two loci can be expressed either as physical or genetic distance.",
      "Although autosomal SNPs are commonly used as genetic markers to infer ancestry or race/ethnicity membership, haploid such as mitochondria, Y-DNA, and X-lined markers are also important to provide separate stories of ancestry of individuals from paternal and maternal sides [42,43].Therefore, genetic structure created due to autosomal markers could be different from those of lineage markers (often influenced by political, social, and migration history of individuals/populations).mitochondrial DNA or mtDNA haploid is the maternally inherited mitochondrial genome (mtDNA) [44].All children inherit mtDNA from their mother, with no admixture from the father.Like Y-line DNA, mtDNA is passed intact from one generation to the next but through maternal line.a) Autosomal DNA (testing both sexes) markers: autosomal DNA tests utilize DNA from the 22 pairs of autosomal chromosomes.Autosomal DNA is inherited from both parents.Autosomal testing provides percentages of ethnicity using autosomal DNA SNP test (i.e., ancestry informative markers), and it is the most commonly used test to infer ancestry across diploid genome.b) Y-DNA or Y-SNPs (paternal line testing) markers: a haploid Y-DNA is the paternally inherited non-recombining portion of the Y chromosome, and it tests only for males.The Y-DNA testing tests the Y chromosome which is passed intact from father to son with no DNA from the mother.Y-DNA testing can then be used to trace direct paternal line.Y-DNA remains the same in each generation, allowing us to compare surname from different regions to see if we are from the same family.Y-line testing does not indicate anything about the contributions of the other ancestors in a family tree.In other words, you could be 3/4th Native American, with only the direct paternal line being European, and this test would tell you nothing at all about those other three Native lines.When testing the Y-chromosome, there are two types of tests, short tandem repeat (STR) and SNP markers.STR tests are best for recent ancestry while SNP tests tell about more ancient ancestry.c) Mitochondrial DNA (maternal line testing) markers:",
      "Additional information about past breeding practices can be gleaned by quantifying the number of reproductive males and females in a population.This can be achieved by comparing levels of genetic diversity between sex chromosomes, autosomes and mtDNA 99 .In cattle, for example, gene flow from aurochs is evident in the autosomes but is absent in mtDNA 41 .This has been interpreted as a management strategy that may have involved allowing insemination of domesticated females by wild bulls 41,100 .In horses, a comparison of the levels of diversity of the Y chromosome and the autosomal chromosomes demonstrated that some cultures allowed fewer males to breed and instead selected specific stallion bloodlines 55 .This male-oriented breeding strategy was not practised by the Romans and only became increasingly prominent in the past 1,000 years as a result of the growing influence of Oriental stallions (Arabian, Persian and Turkmen) 101 .",
      "Dr Ring: What makes the maternal gene so peculiar compared to the paternal?Dr Cookson: If you look in the epidemiologic sense, many studies show that there is increased risk of allergic disease if the mother is affected.However, very few studies have actually set out to test that formally and most of them might suffer from some sort of selection bias because the mother is more likely to be aware of her symptoms and feel guilty, and so on.It is very difficult to explain.Is it genomic imprinting, where the gene is only active when transmitted through the mother?I do not think all of these genes would be imprinted, though it is possible.It also seems that there are effects of the maternal phenotype.The maternal phenotype, if the mother is affected or unaffected, determines the strength of the maternal effect.Again, if a gene was imprinted, you would not expect maternal phenotype to be important.So, I think that this has something to do with maternal/fetal interaction, either through the placenta or shortly after birth.There is the issue of immune conflict between mother and child.At the same time, the mother is trying to prime the infant's immune system.",
      "Genetic and Genomic Discovery Using Family StudiesIngrid B. Borecki, PhD; Michael A. Province, PhD G enetic studies traditionally have been performed on sets of related individuals, that is, families.Mendel's early studies in sweet peas (Pisum sativum) on the inheritance patterns of discrete traits from parents with specific mating types to offspring has shed light on the basic mechanisms of inheritance, including the fundamental laws of segregation of discrete factors (genes) from parents to offspring and the cosegregation of genes that are closely located on a chromosome (linkage).The distribution of traits within families exhibited mathematical segregation ratios in offspring from known mating types.These expected segregation ratios have been used as an important discovery tool in the study of human diseases in pedigrees, providing evidence for a multitude of single-gene disorders.Furthermore, in some cases, trait cosegregation with genetic markers with known positions provides mapping information that enables localization and, ultimately, identification of the relevant causative gene.",
      "In fact, this idea has been pursued before in thecontext of signatures of reproductive isolation and shown to revealpatterns consistent with epistatic gene interactions that arise in theshape of Dobzhansky-Muller incompatibilities [10,11]. In contrast to the mouse data, the available human genotypeswere derived from outbred, ethnically distinct populations. In thiscase pairs of functionally interacting genes can be detectedfollowing a slightly different approach.",
      "Family StructureThe first re-identification method (FAMILY) employs genealogical data accompanying genomic data.Genealogies, rich in depth and structure, permit the construction of complex familial relationships.Consider a simple family structure of two parents and one child.Since the parental genders are guaranteed, there exist 2 variants of this structure, since the child's gender is either male or female.When disease status is taken into account, it is represented as a Boolean variable; either an individual afflicted or not afflicted.In this aspect, all three family members can be represented as three attributes {Father, Mother, Child}, and there exist (father's disease status)*(mother's disease status)*(child's disease status)*(child's gender) = 2*2*2*2 = 16 possible family-disease combinations.In reality, pedigrees are much more robust than a simple nuclear family.For example, a three-generation family of two children per family permits on the order of 10 5 distinct variants of the family-disease structure and 10 6 individuals that could be uniquely characterized.The number of combinationsk is larger when supplementary information, such as living status or medical/genetic features, is considered. 16e ability to determine unique family structures is only one part of the re-identification process.These structures must be linked to identifiable information, which, in many instances, is publicly available in the form of various genealogical databases.These databases are accessible both offline and via the World Wide Web.For example, genealogical records are available in many public databases, including ,Ancestry.com>,,Infospace.com>,,RootsWeb.com>,,GeneaNet.com>,,FamilySearch.org>, and ,Genealogy.com>. {From such data, it is not difficult to construct family structures and, with such information in hand, an adversary can link disease-labeled family structures to named individuals.",
      "Fig. 3. Illustrations of the three CEU pedigrees (black) showing how genetic information from distant patrilineal relatives (arrow; red, patrilineal lines) can identify individuals.Filled squares represent sequenced individuals.To respect the privacy of these families, only abbreviated versions are presented.The sex of the CEU grandchildren was randomized.The numbers of grandchildren are not given.",
      "When I was in high school, I remember often trying to match my friends to their parents at various school functions and being surprised at how easy this was.As human geneticists, in spite of the enormous advances being made in our field, we still cannot answer many of the everyday questions that we are asked, such as: \"Why does he look just like his mother? \"Max Perutz [1], in a recent editorial comment in the New Scientist entitled \"The Molecular Biology of the Future,\" suggested some questions, for, as he put it, \"an examination in some future century. \"Here are two of them: (1) \"The time has come\" the Walrus said, \"To talk of many things ...And why the sea is boiling hot And whether pigs have wings. \"Calculate the amount of genetic information this would require in megacricks.",
      "Using genetic markers, the pattern of inheritance can be tracked throughfamilies. For example, by analyzing a marker linked to the eye color genein several generations, it is possible to determine from which grandparents achild has inherited its eye color alleles. More importantly, nding a markerlinked to a disease can lead to location of the faulty gene causing the disease. Finding the gene is very valuable in the search for the cure. The distance between two loci can be expressed either as physical or genetic distance.",
      "Incontrast, genomic imprinting is due to epigenetic changes withinthe individual causing differential gene expression characterizedby either complete or partial silencing of one parental allele(Barlow, 2011; Abramowitz and Bartolomei, 2012; Ashbrook andHager, 2013). As both mothers and fathers had contact with thepups in our study, our observed PGEs could come from eitherparent. Among quantitative USV traits only peak amplitude of calldisplayed a possible parent-of-origin effect. For call number, callduration, mean peak frequency, and all morphological traits,there were no significant parent-of-origin effect in reciprocalF1 females. In contrast, Thornton et al.",
      "Another way of avoiding stratification is to use family-based samples.This approach has several theoretical advantages: as well as being immune to stratification 114 , these samples can be used to determine whether an allele has different effects on disease when it is inherited maternally or paternally 115 , and DISCORDANT SIB designs [116][117][118] can control for the effects of shared environment.Furthermore, more complex family-based designs are possible 119 that might allow combined association and linkage analysis 120 , and family-based association tests have also been developed for quantitative traits [94][95][96][97][98] .However, pure sibship-based association studies are underpowered relative to case-control studies 107,116,117 , and the requirement for living parents might introduce an age-of-onset bias towards younger patients for diseases that usually arise late in life.Furthermore, family-based samples are often much more difficult to collect, particularly if larger pedigrees are sought.Finally, the most commonly used family-based design, the TRANSMISSION DISEQUILIBIRIUM TEST (TDT; see REF. 114) is susceptible to technical artefacts (see below).",
      "Because mtDNA is not subjected (as far as we know) to sexual recombination and crossover at the time of nuclear meiosis, nature must call on other means to ensure that inevitable germ plasm mtDNA mutations (Medvedev, 1981) are not transmitted.These mutations among primary oocytes, on the face of it, can be expected to increase with time, that is with maternal age.Empirical data on this question are incomplete and conflicting, being mostly confined so far to searches for deletions rather than point mutations (Chen et al., 1995;Keefe et al., 1995).It is inevitable, however, that there will be such mutations and that there must therefore be a reliable physiological mechanism (a) for giving an opportunity for back-mutations to occur, (b) for selecting in favor of those back-mutations (thus preserving the genome) and in favor of rare advantageous mutations, and (c) for preventing the spread of persistent harmful mutations through the population -mutations that are too slight (or too late in origin) to have escaped intraovarian culling.The sheer conservation of the mitochondrial genome over 0.5 billion years or more, despite a mutation rate estimated at 10 -20 times that of nuclear DNA, is ample reason to conclude that such a physiological purification process must exist.",
      "To scrutinize the polygenic networks underlying complex diseases, however, mouse resourcesthat are optimized to study the actions of isolated genetic loci ona fixed background will be insufficient on their own. For example, predisposition to the metabolic syndrome is inherited ina non-Mendelian fashion stressing genetic heterogeneity andmultigenetic pathogenesis (Nandi et al. , 2004). With the reawakening as to the extraordinary genetic resources and phenotypicdiversity archived in extant inbred strains, however, a foundationis in place for tracking down these complex traits and quantitative trait loci (QTL).",
      "Otherwise, tens of thousands or markers will appear significant inthe genome-wise association studies using up to one million geneticmarkers. Approaches to control for stratification include using ofself report of ancestry or genetically derived principle componentsin the analysis. For studies using inbred mouse lines, a cladogramwhich is a hierarchical grouping based on phylogenetic analysis ofstrain relatedness can be created to subdivide inbred strains intomore genetically homogenous subgroups.",
      "Although bilateral descent is the norm in Western societies, it is not universal and there is variation with cultural practices around lineage.In certain societies, individuals place greater importance on (and have greater knowledge about) one side of the family than another (unilineal descent).Thus, individuals in patrilineal groups trace relationships through males only so that your father's brother's children are members of your family, but not your father's sisters (Kottak, 2007).They are members of their husband's group or family.Efforts to create a family pedigree may be hampered if the participant is not familiar with her mother's relatives, but her mother's brother's children (her cousins) may be able to supplement her overall family history.Knowledge about the cultural system of unilineal descent avoids assuming the universality of bilateral descent.Cultural beliefs such as these also have implications in the conduct of genetic research in terms of confidentiality and autonomy (Benkendorf et al., 1997;Wertz, 1997).One cannot assume that the named proband is in a position to speak for the extended family in agreeing to participate in any genetic research (DudokdeWit et al., 1997).",
      "In particular in polygynous species, a femalesoffspring may have different fathers and are thus more closely related through the maternalthan the paternal line. Therefore, any fitness cost to mothers, such as increased provisioningand care, affect maternally derived genes more strongly than paternally derived genes,leading to the silencing of the maternal copy (i.e. paternal expression) of genes that increaseresource transfer. 5. Coadaptation between offspring and maternal traitsThe genetics of the co-evolution of parental and offspring traits has been investigated usingquantitative genetics models and in several empirical studies (Agrawal et al.In thisscenario, genes expressed in parents will be selected for their effects on parental behaviourwhile genes expressed in offspring will be selected for their effects on influencing parentalbehaviour. At the genetic level the predicted conflict between paternal and maternal genomes isthought to have led to the evolution of genomic imprinting (monoallelic gene expression). Genomic imprinting effects are good examples of offspring genetic effects on maternal carebecause of the impact on the quality of maternal care and level of resource provisioning (e.g. Li et al. , 1999)."
    ],
    [
      "When a cell divides in two, both daughter cells must receive a copy of allthe DNA, i.e. the whole genome. During replication the two strands of themother cell DNA are separated, and new nucleotides are put together to maketwo double helices identical to the original one, see Figure 2.1. ATAAGACCG. . . . . . . . ATTCTGGCGACCG. . . . TGGCTA. . ATTCCG. . . CTGGCFigure 2.1: A DNA chain consists of two strands of complementary nucleotides.WhenDNA is replicated, two double chains identical to the original one are created. The human genome consists of approximately 3 billion nucleotide pairs. The chain is divided into pieces called chromosomes. A gene is a short segment of a chromosome where the nucleotide sequence gives the blueprint fora particular substance in the body, for example insulin. Only a small fractionof the DNA consists of genes. In between the genes there are long non-codingregions of which the function is largely unknown.Germ cells originate from 46-chromosomecells, and a sophisticated process called meiosis ensures that exactly 23 chromosomes, and exactly one from each homologous pair, ends up in each daughter cell. Before the homologous chromosomes are distributed to the daughtercells they are paired up side by side. While they are positioned close togethera process called crossover often occurs, see Figure 2.2. The homologous chromosomes randomly exchange large chunks of DNA. As a result, each chromosome that a child has inherited from a parent will most often contain segmentsfrom both grandparents.",
      "Replication handlingReplication is a significant part of any comparative experimentation to raise accuracy and more significantly to deliver a basis for recognized statistical interpretation which is nowadays becoming broadly accepted for genomic data.In genetic and genomic context, replication can have various forms [97]: technical replicates, duplicate gene spots, and biological replicates.It is vital to understand that any sort of replication provides information only concerning the specific source of changeability related to that kind of replication and no other.Based on the experimental setting, it may consequently be imperative to consider one, two or all these categories of replicates.",
      "Central dogma-An explanation of the flow of genetic information within a cell.Information is stored in the DNA of the genome, transcribed into RNA, and translated into protein.With a few exceptions, genetic information follows this path only in the forward direction.Basics of Molecular BiologyDeoxyribonucleic acid (DNA) is a molecule with 2 strands that are wrapped around each other in a helical formation, hence its description as a double helix (Figure 1).The outer portion of the helix contains the sugar and phosphate backbone; the inner portion contains the coding bases: adenine (A), cytosine (C), guanine (G), and thymine (T).The genetic information of an organism is determined by the order of the sequence of the bases; with 4 bases available, the number of potential sequences is almost infinite.The versatility of DNA results from the obligatory pairing of bases in the 2 strands, forming base pairs.An adenine in 1 strand is always matched up with a thymine in the other strand, and cytosine is always paired with guanine.Thus, the 2 strands contain redundant information, and each can serve as a template on which a new complementary strand can be synthesized.This allows easy duplication of the DNA so that, when a cell divides into 2 cells, each descendant cell receives the same genetic information as the original cell.Figure 1.The structure of DNA.Each DNA strand has a sugarphosphate backbone (not shown in detail) with a sequence of bases that come in 4 versions: adenine (A), cytosine (C), guanine (G), and thymine (T).Two DNA strands can combine to form a double helix, the stable form of DNA found in chromosomes.Holding the strands together are base pairs: Guanine on 1 strand binds to cytosine on the other strand, and adenine on 1 strand binds to thymine on the other strand.Thus, the 2 strands are complementary and contain redundant information.Figure 8. Meiosis, part 1.Before the first cell division, meiotic recombination (crossing over) between a chromosome pair occurs.Figure 9. Meiosis, part 2. The second cell division yields gametes, which have only half of the complete genome (unpaired chromosomes).Two gametes subsequently fuse (fertilization) to create a zygote that has a complete genome and can give rise to an organism.Figure 8. Meiosis, part 1.Before the first cell division, meiotic recombination (crossing over) between a chromosome pair occurs.Figure 9. Meiosis, part 2. The second cell division yields gametes, which have only half of the complete genome (unpaired chromosomes).Two gametes subsequently fuse (fertilization) to create a zygote that has a complete genome and can give rise to an organism.",
      "When a cell divides in two, both daughter cells must receive a copy of allthe DNA, i.e. the whole genome. During replication the two strands of themother cell DNA are separated, and new nucleotides are put together to maketwo double helices identical to the original one, see Figure 2.1. ATAAGACCG. . . . . . . . ATTCTGGCGACCG. . . . TGGCTA. . ATTCCG. . . CTGGCFigure 2.1: A DNA chain consists of two strands of complementary nucleotides.WhenDNA is replicated, two double chains identical to the original one are created. The human genome consists of approximately 3 billion nucleotide pairs. The chain is divided into pieces called chromosomes. A gene is a short segment of a chromosome where the nucleotide sequence gives the blueprint fora particular substance in the body, for example insulin. Only a small fractionof the DNA consists of genes. In between the genes there are long non-codingregions of which the function is largely unknown.",
      ". . . . . . . Appendices301Appendix ASummaryAll organisms have a genome made of DNA (deoxyribonucleic acid). The genome can be found in nearly every cell and is the blueprint for thegrowth, development, maintenance and repair of the body. It performsthese functions by transcribing small pieces of DNA, the genes, fromthe genome and translating them to proteins. These proteins are thetiny workhorses of the body that break down food, give bones theirstrength, make muscles move, let brains think, and so on.",
      "Every nucleated cell in our body, with the exception of egg and sperm, has a complete genome in its nucleus.Each time the cell divides by the process of mitosis, all the DNA in that cell is replicated, so that each of the two new daughter cells has its own copy of the entire genome.The mitochondria, which produce the energy required for all the cell's functions, contain a small circular DNA molecule that is also part of the genome.Every living organism has a complete genome in each of its cells.And the structure of all DNA is the same.The DNA in human cells has the same structure as the DNA in the cells of a butterfly, a whale, a flower, or a worm.What differs is simply the amount of DNA carried by each organism and the order of the nucleotides in each strand.",
      "IntroduclJonEver since the structure of DNA was elucidated by Watson and Crick in 1953, it has been generally assumed that genomic DNA, in view of its vital role in transferring hereditary information from generation to generation, is a stable molecule unaltered in its structure by the surrounding events.This taken for granted, its remarkable attribute of stability has turned out to be a myth.As noted by Haynes (1988) DNA is made up of rather ordinary molecules that are not endowed with any peculiar kind of quantum mechanical stability.As such, DNA must be able to undergo all kinds of structural modifications at the body temperature and with many other chemicals in proximity.Much evidence has accumulated in recent years to prove that this is indeed the case, and normal cellular metabolism itself is enough to cause various types of damage to the genomic apparatus.If the genomic DNA can be assaulted in so many ways the natural question that would emerge is: How is genetic informational integrity maintained and transmitted through generations?",
      "The second form of genome partitioning was by DNA replication direction.Since the entire genome is replicated every time a cell divides (but only a portion is transcribed), replication direction has the potential to exert larger asymmetries in mutational data.However, determining direction is much more challenging for replication than transcription, since the precise locations of replication origins in the human genome are not known.This has precluded a comprehensive analysis of replicative strand asymmetry thus far.",
      "Each gene is a segment of deoxyribonucleicacid (DNA) and the genes are joined together to make up a set of very long DNA moleculescalled chromosomes. In diploid organisms like humans and mouse, there are two copies of eachchromosome. One copy is inherited from each parent. DNA is comprised of a sequence of nucleotides and the four primary DNA bases found innucleotides are Adenine(A), Cytosine(C), Guanine(G), and Thymine(T). Each base binds withanother specific base (T with A and C with G).",
      "If this DNA were to be uncoiled and laid out end toend, it would extend about 3 m. Obviously, this cannot possibly fit into a cell,and extended DNA would be susceptible to breakage during replication andcell division. In eukaryotes, genetic material is thus organized into complexesof DNA with core histones and other chromosomal proteins that together formchromatin. The chromatin repeating unit includes two copies each of four corehistones H2A, H2B, H3 and H4 (collective molecular mass 206,000) wrappedby 146 bp of DNA.",
      "The core of the human genome is a DNAdouble helix containing ~3 billion base pairsof genetic information. It is continuously challenged by a variety of genotoxic stresses thatcause ruptures of the DNA sugar-phosphatebackbone. DSBs are the most lethal type ofDNA damage. They can be caused by collapseof the DNA replication fork or, less commonly,induced directly by environmental insults suchas ionizing radiation or radiomimetic drugs. To manage these lesions, cells have evolved twomain pathways of DSB repair. Homologousrecombination occurs in mitotic cells, usuallyduring the S and G2 phases.",
      "Cellular and Genetic ChangesThis section will explain how cells normally divide.It will also describe how an unexpected change in the structure of DNA can sometimes cause harm to the body.New tools to study genetic variations of common diseases and to identify genetic variations common to specific diseases will also be presented.Table 1. -Glossary of Genetic and Genomic TermsDeoxyribonucleic acid (DNA) -The chemical inside the nucleus of a cell that carries genetic instructions for making living organisms.Double Helix -The structural arrangement of DNA, which looks something like an immensely long ladder twisted into a helix or coil.The sides of the \"ladder\" are formed by a backbone of sugar and phosphate molecules, and the \"rungs\" consist of nucleotide bases joined weakly in the middle by hydrogen bonds."
    ],
    [
      "Gene editing has gained considerable interest with the identification of the CRISPR-Cas9 system, 27 which allows for a targeted modification in the DNA sequence of an organism.Researchers can utilize their knowledge of the basic biology of the gene and its protein function to precisely change the DNA sequence, thus altering the protein function of the gene and allowing for edits to stay within the species.Researchers at the University of Missouri used the CRISPR-Cas9 system to modify the CD163 gene such that the PRRS virus is not able to replicate inside the pig. 28This slight modification of the swine genome through gene editing keeps the pigs from succumbing to PRRS which has an annual estimated loss to the United States swine industry of over $660 million per year.Despite this benefit, given the public's concerns over food safety, it is likely that approval for such technology is years away in the US, Canada and Europe.However, in some cultures, there is a wide range of non-livestock species that are consumed.Therefore, it is conceivable that these countries and cultures may be open to transgenic/gene edited livestock.They may see the importance of useful gene editing which may lead to approval and consumption of reasonable genetically edited animal products such as those with modifications that are already found in nature or those that offer a substantial welfare benefit to society.",
      "As a researcher who has devoted an entire career since 1994 to the development of genome editing tools and methods, I have been amazed by the rapid progress in the field over the last few years.Considering the widespread use of the tools, I am sure that the pace will continue to accelerate.Indeed, programmable nucleases, may eventually enable humans-products of evolution-to become masters of evolution.delivered preassembled recombinant Cas9-guide RNA ribonucleoproteins (RNPs) into animal embryos 6,9 and plant 11 and mammalian cells [73][74][75] .Indeed, Cas9 RNPs were rapidly turned over in cells 73 , reducing off-target effects and mosaicism in gene-edited organisms 11 .Cas9 RNPs can be delivered into cells by various methods, including microinjection 6,9 , electroporation 73 , lipofection 74 and protein transduction 75 .Importantly-and unlike in conventional gene therapy, where therapeutic genes are delivered via plasmids or viral vectors-Cas9 RNP delivery does not involve the use of exogenous DNA; host innate immune responses against foreign DNA are not elicited, and undesired integration of foreign DNA into the host genome is avoided.",
      "In comparison to a transgenic approach, a gene editing technique such as CRISPR-Cas9 offers the advantage that gene-edited crops are not considered genetically modified organism (GMO) in some countries, such as the US, where the demand for natural food colorants such as anthocyanins is high.Indeed, the use of GMO crops as a source of natural pigments may be inconsistent with consumer interests.However, carrot cultivars engineered with either the transgenic or gene editing approach have not been reported so far, but their development is possible.",
      "The notable accuracy and versatility of CRISPR-Cas for genome editing also opened the door to its use in preclinical and translational settings.In the latter case, CRISPR in vivo gene editing has led to several proof-of-concept studies that would have been unachievable without it, as in the first ever correction of inherited pathogenic mutations linked to degenerative disease in a living organism [22] and even shown to be possible in human embryos [23,24].It also has great potential in the field of precision medicine as large-scale population DNA sequencing studies have provided vast amounts of information linking particular diseases with specific genetic mutations which could, in theory, be targeted through CRISPR [25,26].This could be used during the identification and validation of potential DNA targets during the development of personalised drug or cell therapies, which will require the generation of engineered cell lines and/or animal models.Techniques such as HDR-mediated gene targeting are too labour intensive, with low targeting efficiencies and long times necessary for their establishment, and consequently are not ideally suited for drug discovery purposes.Conversely, CRISPR-Cas has been proven to be efficient for editing virtually any kind of cell line, from primary immune cells to induced pluripotent stem cells (iPSCs) [27,28].Additionally, CRISPR can also be used for functional screening in the development of combined inhibitory therapy aimed at strengthening the efficiency of targeted therapeutics.An example of the latter is shown in a study where a variation of the technology known as CRISPR interference (CRISPRi) was used in genome-wide scale to identify different survival pathways used by cancer cells after oncogene inactivation and allowing the identification of successful combination therapies [29].In terms of translational applications, the overall safety of CRISPR genome editing in humans will require long-term scrutiny before its adoption in the clinic.Nonetheless, a number of CRISPR-based clinical trials are currently in progress, including studies focused on targeting patients' own T cells in order to improve the immune response towards some forms of malignant cancer [30,31], and others aimed at correcting pathogenic mutations in the hematopoietic cells of patients with beta-thalassemia and sickle cell disease [32].Caveats and Ethical Concerns of CRISPR-Cas ApplicationsDespite the presence of both a PAM sequence and a specific gRNA, the CRISPR-Cas9 system is not infallible.In fact, DSBs can occur at different sites in the genome, potentially causing so-called \"off-target\" effects.This eventuality remains to date the biggest concern in the field, as possible undesirable modifications must be properly identified and followed in order to guarantee safety for medical purposes.Nevertheless, there is still little evidence of the biological consequence of Cas9 off-target effects.Two recent studies describe new methods to investigate potential off-target effects in both mammals and plants [33,34].In both cases, whole-genome sequencing revealed that selective nucleotide changes, such as conversion of an adenine to a guanine, caused off-target occurrence very rarely, with a frequency comparable to the one of spontaneous mutations.However, substitution of a cytosine with a thymidine was linked to a sizable number of off-target mutations.This newly acquired information adds to the plethora of studies conducted on the safety of CRISPR, which altogether highlight the need for the establishment of clinical standards for the future use of genome-editing techniques in the clinic.Despite this and other technical challenges still ahead for CRISPR genome editing, the pace at which this technology has developed in recent years suggests many of these concerns could be addressed soon, as long as proper ethical guidelines and regulatory mechanisms are established.ConclusionsThere is no reason to doubt that the development of CRISPR-Cas genome editing represents an unprecedented breakthrough in modern science, as it has potential applications in a wide array of disciplines ranging from agriculture, zoology and renewable energy to biomedicine and synthetic biology.This powerful tool holds promise for further elucidating the molecular causes of ageing by allowing scientists to probe genetic and epigenetic pathways with a level of sophistication that was unattainable just a few years ago.It will allow so in traditional animal and cell models of ageing, but it will also drastically accelerate the generation of refined versions of those models or even allow the development of new research approaches in non-model organisms.Moreover, CRISPR-based genome editing is already having a significant impact in research aiming to understand the cellular and molecular origins of age-related diseases, as well as developing potential treatments against them.The application of CRISPR-Cas gene editing for the treatment of age-related diseases is not over the horizon yet, as it will require the identification of causative genes and their role under a variety of contexts that could be as diverse as the ageing process is across individuals.However, CRISPR-Cas might also hold the key for solving such conundrum, as it has opened the way for achieving true personalised medicine by providing both the precision and scalability required for conducting genome-wide functional screens during the refinement of drug-and cell-based therapies for age-related diseases.Since its discovery, CRISPR-Cas technology has ignited a biological revolution by providing a highly versatile platform that allows fast and efficient genome editing in an ever-growing list of organisms.In this chapter we will first describe the most recent advances in the development and application of the CRISPR-Cas platform in biomedical research.Then we will discuss the most recent and notable basic research applications of this technology in the study of the molecular causes of ageing.Finally, we will review how CRISPR-Cas has been used for creating new models for the study of age-related diseases, as well as for manipulating diseaseassociated gene pathways.",
      "Caveats of advanced genome editing toolsOff-target effects.The DNA-binding domains of ZFNs and TALENs need to be very specific for the target site to avoid off-target cleavage, which results in unwanted mutations and potentially cytotoxic effects [27].CRISPR/Cas9 is also known to generate off-target alterations, albeit apparently at low incidence [28,29], since mispairing is allowed between the guide RNA and the genomic DNA.Nonetheless, caution is required in their design and use.Some strategies involving the optimization of the guide RNA/Cas9 include using of software tools to predict potential off-target sites (http://omictools.com/crispr-cas9-Figure1: Genome editing methodologies which can be applied to human pluripotent stem cells.Homologous recombination (HR), or the more advanced tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system can be applied to human pluripotent stem cells (hPSCs) either to 1) create naturally occurring mutations or 2) repair a mutation to generate isogenic controls in hPSCs, to understand the function of a gene of interest.c1268-p1.html),truncating the guide RNA (<20 nucleotides) to decrease off-target mutagenesis [30], lowering the dosage of guide RNA and Cas9 plasmids, and decreasing the number of mismatches between the guide RNA and the genomic DNA.A \"double nick\" system with Cas9 nickase, which contains a single inactive catalytic domain, may also be used [31e33].",
      "CRISPR screening technologiesThe discovery of CRISPR-Cas9 as a sequence-specific programmable nuclease democratized gene editing and fueled progress in forward genetic screening [20 , 66] .Genetic screens using Cas9 with a pooled singleguide RNA (sgRNA) library allow the interrogation of seemingly all genes in a genome in a single experiment [96 , 97] [null] .Engineered Cas9 variants further extend the versatility of forward genetic screening.Catalytically inactive Cas9 (dCas9) fused with chromatin effector domains permit specific activation (CRISPRa) or inhibition (CRISPRi) of gene expression [37 , 54] .Recently developed and emerging technologies -base editors, prime editors, and Cas transposases -are beginning to enable new types of genetic screens with directed, controlled, and on demand mutations by allowing the creation of user specified modifications, such as single base conversion, deletions, and insertions [4 , 42 , 58] .",
      "Coming on the heels of engineered nucleases, CRISPR-Cas9 tools have accelerated the pace of genomic research by permitting highly efficient knockouts or edits of virtually any gene in cells or model organisms.Multiple CRISPR-Cas9-based clinical trials are in progress or are expected to begin soon.Although Cas9engineered cells haven't yet demonstrated efficacy at scale, early trial results suggest that such cells are stable and don't cause acute adverse reactions in humans.Long-term safety is yet to be determined.Current applications largely focus on single-gene disorders for which gene editing can be carried out ex vivo on appropriate cells, such as bone marrow hematopoietic stem cells in the case of sickle cell anemia.Exploration is under way to develop delivery systems that can target the gene-editing apparatus to the appropriate tissue in vivo.Over the past 8 years, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) technologies have emerged as accessible and adaptable tools for studying and altering genomes. 5RISPR-Cas9 can be used to induce genome edits by creating targeted DNA breaks that trigger site-specific DNA repair.In nextgeneration formats, it can also control the transcriptional output of genes or alter genome sequences using a process of nucleotide base editing that does not require repair of DNA breaks.As these technologies continue to mature, it will become increasingly possible to alter cellular genomes efficiently and accurately.",
      "The type II CRISPR-Cas9 systems, repurposed from prokaryotic adaptive immune responses, are now widely used for targeted genome modifications in plants, animals, and human cells (Kim et al. 2014;Woo et al. 2015;Zuris et al. 2015).In particular, Cas9 nucleases have shown promise for gene and cell therapy (Maeder and Gersbach 2016).Typically, these nucleases are expressed or delivered in vivo using plasmid DNA or viruses (Yin et al. 2014;Ran et al. 2015).However, plasmid DNA delivery is often inefficient, especially in vivo, and can cause integration of small plasmid fragments degraded by endogenous nucleases at on-target and offtarget sites in the genome (Kim et al. 2014).Viral delivery of Cas9 can be highly efficient in vivo (Ran et al. 2015;Long et al. 2016;Nelson et al. 2016;Tabebordbar et al. 2016), but may be hampered by antibodies or T cells induced against the protein (Shankar et al. 2007;Calcedo et al. 2015;Chew et al. 2016).We and others have shown that preassembled Cas9 ribonucleoproteins (RNPs) can be delivered to human primary and stem cells and mice to modify target genes (Kim et al. 2014;Schumann et al. 2015;Zuris et al. 2015).Cas9 RNPs are rapidly turned over in cells, reducing off-target effects.Furthermore, Cas9 RNPs are unlikely to be limited by host immune systems because they function and disappear before the generation of antibodies and T cells directed against them.Currently, despite these advantages of RNPs, the difficult delivery of Cas9 RNPs in vivo limits its utility for therapeutic applications (Zuris et al. 2015).Here, we show that in vivo genome editing of an wild-type gene, whose up-regulation is responsible for pathogenesis, could be a new therapeutic modality for the treatment of nongenetic degenerative diseases.Our ultimate goal is to harness Cas9 RNPs for a clinical application of therapeutic genome surgery in patients with AMD.",
      "Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases have revolutionized the field of gene editing and have tremendous application in the field of molecular medicine [98][99][100][101][102].Despite a significant surge in CRISPR/Cas9mediated genome editing in various disease models, the progress in the field of AD has lagged behind substantially.We believe that genome editing can significantly improve the development of AD models and also create novel opportunities for the development of the next generation precision targeted AD gene and stem cell therapies.Since there are several excellent review articles on CRISPR/Cas9-mediated genome editing, here we will limit our focus on select recent articles that are noteworthy.CRISPR/Cas9 system can be engineered to either activate transcription (gain-of-function) or achieve gene silencing (Loss-of-function).Dahlman et al. have developed a CRISPR-based system that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to activate and knockout different genes in the same cell [103].Konermann et al. have used structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci [104].Using crystallographic studies, they have engineered a combination of sgRNA2.0,NLS-dCas9-VP64 and MS2-p65-HSF1 to develop one of the most effective transcription activation system.",
      "Limitations of CRISPR-Cas9CRISPR provides a simple and easy tool not only for in vitro use but potentially also for in vivo genome editing.However, there are limitations and downsides to this approach.First, and despite considerable improvements in the technology, the risk of the offtarget effect remains and must be considered carefully.Second, DSB may lead to wide-ranging deletions or recombination events involving the on-target site (204).Third, in cycling cells, DNA double strand breaks caused by Cas9 cleavage may trigger a P53 response leading to apoptosis and enrichment for potentially oncogenic P53-deficient cells (205,206).Fourth, subjects may generate antibodies to Cas9, potentially limiting gene therapies (207,208).Genome editing tools that target the desired genomic region and allow for variants to be altered (e.g. from risk to protective), or for more substantial changes to be made (e.g. the deletion of a longer stretch of DNA harbouring a number of variants) and can help to answer each of these questions.These technologies are evolving rapidly (Figure 1 and Table 2).The most recently developed of these, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, originally developed by Doudna, Charpentier and their colleagues (72,73) and Zhang and his colleagues (50) has become a widely used tool for this purpose.Engineered CRISPR/Cas9 technology uses a guide RNA (gRNA) to direct CRISPR-associated endonuclease (Cas) to the target DNA and generate a double strand DNA break.Correction of a mutation or variant in the target DNA sequence can then be carried out by homology-directed DNA repair (HDR) with a donor template.Since its discovery eight years ago, CRISPR technology has evolved quickly to be a critical part of the molecular biologist's toolbox.",
      "INTRODUCTIONGenome editing technologies based on the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 enable rapid and efficient modification of endogenous genes in a variety of cell types, allowing for analysis of gene function in many organs in vivo.CRISPR-Cas9 induces DNA double strand breaks (DSBs) at single-guide RNA (sgRNA)-specific loci in the genome, which are repaired through either non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways.While NHEJ introduces unpredictable pattern of insertion or deletion (indel) mutations, HDR directs a precise recombination event between a homologous DNA donor template and the damaged DNA site (Cong et al., 2013;Cox et al., 2015;Doudna and Charpentier, 2014;Heidenreich and Zhang, 2016;Jinek et al., 2012;Mali et al., 2013;Sander and Joung, 2014;Wang et al., 2013;Yang et al., 2013).Thus, HDR can be used to precisely introduce sequence insertions, deletions or mutations by encoding the desired changes in the donor template DNA.",
      "CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development.What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA.Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions.For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene.In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening.In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development.What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA.Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions.For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene.In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening.In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.The recent development of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 for experimental purposes has dismantled the perception that genome editing technology is off-limits for screening in mammalian systems (Heintze et al., 2013).Since this system employs the basic principle of Watson-Crick base pairing for gene targeting, generation of libraries with whole-genome target coverage is relatively easy and cost-effective.For instance, simple protocols are available to synthesize pooled lentiviral libraries by in silico design of oligonucleotides, which can then be cloned, packaged and delivered to cells by viral transduction (Paddison et al., 2004;LeProust et al., 2010).Similarly, the generation of arrayed libraries can be achieved by following protocols originally developed for arrayed shRNA library production that have been in use for a number of years (Moffat et al., 2006).All in all, the stage is set for CRISPR to make an enormous impact on genomic screening and thus scientific discovery in the coming years, and recent demonstrations of this system have shown great promise (Shalem et al., 2015).However, a number of technical challenges must be addressed in order to maximize the benefit of this technology.In this review, we will discuss current applications of CRISPR in functional genomics and provide a perspective on future developments in this area.",
      "Genome editing for crop improvementReports of CRISPR-Cas9-based genome editing first appeared in 2013 (Cong et al., 2013;Feng et al., 2013;Mao et al., 2013).Since then, genome editing technologies have proven to be powerful and efficient tools for the improvement of many crop species.At present, genome editing has been widely used to introduce/modify agronomically important traits, such as increased yield, improved nutritional quality, and resistance to biotic and abiotic stresses, in multiple crops, including rice, wheat, maize, tomato, and potato (Lu et al., 2017;Soyk et al., 2017;Tang et al., 2017;D'Ambrosio et al., 2018;Ye et al., 2018;Miao et al., 2019;Zhang et al., 2019;Zhong et al., 2019;Butt et al., 2020;Zhang et al., 2020c;Li et al., 2021b;Zhan et al., 2021).CRISPR-Cas-based genome editing has been extended to targeted mutagenesis, base editing, and precisely targeted gene/allele replacement or tagging in plants.mportantly, using CRISPR-Cas9 technology, transgenes present in the genomes of genome-edited plants can be removed by chromosomal segregation via a simple self-pollination or hybridization step.Gene editing technologies continue to be developed and utilized (Mao et al., 2013;Lu and Zhu, 2017;Lu et al., 2020)."
    ],
    [
      "Second, and perhaps moreimportant, is the difference in the size and types of thegenetic reference populations. In our previous study, wemapped the QTL with 36 F2 mice that were genotyped at82 markers. In the current study, by comparison, we wereable to map QTLs after examining 342 mice from 55 strainsthat were genotyped at approximately 4000 markers.",
      "This contrast can be exploited to identify subregions that underlie the trans-QTLs [67]. SNPs were counted for all four pairs of parental haplotypesBvs D, B vs H, B vs C, and L vs Sand SNP profiles for the fourcrosses were compared (figure 6). Qrr1 is a highly polymorphicPLoS Genetics | www.plosgenetics.org8November 2008 | Volume 4 | Issue 11 | e1000260QTL Hotspot on Mouse Distal Chromosome 1Figure 5. QTL for aminoacyl-tRNA synthetases in distal Qrr1.",
      "The traditional approach to QTL mapping is to usetwo strains that differ maximally in the phenotype asparental strains for genetic crosses, with the followingcaveats. QTL analysis based on a single cross will mostlikely reflect only a small portion of the net geneticvariation, and QTL detection will be limited to regionswhere the two progenitor strains have functional polymorphisms. Data from multiple crosses, or from an HS,will overcome this limitation and can also be used toreduce QTL intervals [5,30].",
      "These candidate genes are then sequenced in the two parental inbredstrains looking for sequence dierences in coding or regulatory regions. After ne mapping the QTL interval and shortening the list of plausiblecandidate polymorphisms, the major challenge remains \u0001 proving denitivelywhich nucleotide polymorphism underlies the QTL. The most direct proofwould be replacing one strains allele with another strains allele (creating aFIG. 1. Intercross breeding strategy for mapping quantitative trait loci (QTLs). On the right, the parental, F1 hybrid, and intercross (F2) mousegenerations are depicted.",
      "Furthermore, splicing QTLs(sQTLs) rather than eQTLs could comprise the molecular mechanism linking DNA variants with YFP53; thus, sQTL analysis could uncover genes that would not normally bedetected at the level of differential gene expression (DGE),53 and thus, a differentially181182Molecular-Genetic and Statistical Techniques for Behavioral and Neural ResearchFigure 8.5 Schematic for immediate, rapid ne mapping in select F2 recombinants of the RCC-F2cross. Top panel: Genome-wide signicant QTL (green trace; red dashed line  signicance threshold;blue vertical lines  Bayes credible interval).",
      "The fuzzy functional boundaries of genes andthe high density of sequence variants in linkage disequilibrium shifts the burden of prooffrom pure mapping to functional genomics, comparative analysis of human cohorts,complementary animal models, and direct pharmacological and genetic engineering (Smemoet al. , 2014). Author ManuscriptMapping with the BXDs has high powerHow many replicates and strains are needed to detect and resolve QTLs? To start with theconclusionit is almost always better to study small numbers of as many strains as possible(Andreux et al. , 2012; Belknap, 1998).",
      "Interval-specific haplotype analysisApproximately 97% of the genetic variation betweeninbred mouse strains is ancestral [22], so regions ofidentity by descent (IBD) between two strains used todetect a QTL are highly unlikely to contain the causalgenetic polymorphism underlying the QTL [28]. Forexample, a cross between C57BL/6J and A/J mice detectedwww.sciencedirect.coma blood pressure QTL on Chr 1 [7].",
      "Interval-specific haplotype analysisApproximately 97% of the genetic variation betweeninbred mouse strains is ancestral [22], so regions ofidentity by descent (IBD) between two strains used todetect a QTL are highly unlikely to contain the causalgenetic polymorphism underlying the QTL [28]. Forexample, a cross between C57BL/6J and A/J mice detectedwww.sciencedirect.coma blood pressure QTL on Chr 1 [7].",
      "At present, the BXD panel is composed of 80 different strains that all have beenfully genotyped.26 Variation in any quantifiable trait can be associated with thesegregation of parental alleles, and linkage genetics can map this variation toquantitative trait loci (QTLs), thereby identifying the genomic region(s) affectingthat trait. An overview of the QTL mapping approach is depicted in Figure 2. Classical QTL analysis has permitted the identification of loci that areassociated with variation in HSC traits.",
      "The progenitor mouse strainsshould have sufficient variation for the traits of interest and they should be genetically diverseenough to enable genetic mapping (BENNETT et al. 2006; FLINT 2003; GRISEL 2000). Thesample size required for the identification of QTL depends largely on the effect size that aQTL contributes to phenotypes on interest. Inference about QTL can be made if one or moregenetic markers are over- or underrepresented in the analysed individuals. Genotyping isoften done by means of microsatellite markers, which contains mono, di-, tri-, ortetranucleotide tandem repeats flanked by specific sequences (Figure 4a).This comparison gives information about the reliability of the observed genotypeinformation: The more the marker locations differ between the two maps (which signifiesvariation in marker positions), the higher the possibility of genotyping errors. QTL mapping was done in several stages to identify loci acting individually and QTL thatinteracted, either additively or epistatically. To determine individually-acting QTL, a singleQTL genome scan was conducted with the function scanone.In general,linking genetic variation with trait variation identifies QTL and a significant linkage ofphenotype and genotype suggest that the DNA status helps to determine trait expression. As stated above, mouse QTL studies provide distinct advantages over human studiesin the examination of genetic causes of a quantitative trait (e.g. alcoholism), even in theabsence of specific hypotheses regarding its aetiology or candidate genes.",
      "Importantly, whereasthese studies required substantial labor, time, and resources, X-QTL is a quick and easyapproach to achieve a comparable level of genetic dissection. The levels of complexityobserved here (e.g. 14 loci explaining 70% of the genetic variance for 4-NQO resistance) arestill dramatically lower than those seen in for some human traits in GWAS (e.g. 40 lociexplaining 5% of the variance for height 2,5). One obvious explanation is the difference inexperimental designs (line crosses vs. population association studies), but differences ingenetic architectures among species and traits may also contribute.",
      "The method uses two pieces of information: mapping data from crosses thatinvolve more than two inbred strains and sequence variants in the progenitor strains within the intervalcontaining a quantitative trait locus (QTL). By testing whether the strain distribution pattern in the progenitor strains is consistent with the observed genetic effect of the QTL we can assign a probability that anysequence variant is a quantitative trait nucleotide (QTN). It is not necessary to genotype the animals exceptat a skeleton of markers; the genotypes at all other polymorphisms are estimated by a multipoint analysis.",
      "The method uses two pieces of information: mapping data from crosses thatinvolve more than two inbred strains and sequence variants in the progenitor strains within the intervalcontaining a quantitative trait locus (QTL). By testing whether the strain distribution pattern in the progenitor strains is consistent with the observed genetic effect of the QTL we can assign a probability that anysequence variant is a quantitative trait nucleotide (QTN). It is not necessary to genotype the animals exceptat a skeleton of markers; the genotypes at all other polymorphisms are estimated by a multipoint analysis.",
      "Genotyping all the individual progeny formarkers that show allelic variation between the parental strains (either single nucleotide polymorphisms or simple sequence repeats) will allow the detection of associations between trait values and marker genotype, and in this way demonstrate to whichset of markers a QTL is linked. To reduce the genotyping effort, selective genotypingof the individuals at the extremes of the phenotypic spectrum can be performed (20,23). Although these three approaches are in general considered to be the best to detect andmap QTL, they have several disadvantages for quantitative traits involving HSC.",
      "So, how do you go about planning and performing a QTL study, and howdo you identify the responsible gene within a QTL that you have identified? Generally, one starts by performing a strain survey to find two parental inbredstrains that have a markedly different trait. One can now look up many differenttraits of inbred mice online at the Mouse Phenome Database (http://phenome. jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home). However, the trait you maywant to study may not be present in wild type mice, so you may want to crossa mutant (or genetically engineered) strain onto several inbred strains.",
      "In any case, precision much finerthan this, while welcome, will often not be critical. The fuzzy functional boundaries of genes and the high density of sequence variants in linkage disequilibrium shifts the burden of proof frompure mapping to functional genomics, comparative analysis ofhuman cohorts, complementary animal models, and direct pharmacological and genetic engineering (Smemo et al. , 2014). Mapping with BXDs has high powerHow many replicates and strains are needed to detect andresolve QTLs?",
      "These candidate genes are then sequenced in the two parental inbredstrains looking for sequence dierences in coding or regulatory regions. After ne mapping the QTL interval and shortening the list of plausiblecandidate polymorphisms, the major challenge remains \u0001 proving denitivelywhich nucleotide polymorphism underlies the QTL. The most direct proofwould be replacing one strains allele with another strains allele (creating aFIG. 1. Intercross breeding strategy for mapping quantitative trait loci (QTLs). On the right, the parental, F1 hybrid, and intercross (F2) mousegenerations are depicted.",
      "QTL mapping studies thenseek to detect the polymorphisms underlying the complex traits of interest byscanning for alleles that co-vary withthe traits. Similar experiments also can be conducted with special derivatives of inbredstrains known as recombinant inbred(RI) mice. These animals are derivedby cross-breeding two or more distinctparental strains (which often divergewidely for the trait of interest), followedby inbreeding of the offspring for severalgenerations (Bailey 1971). Given thecorrect breeding strategy, this method1This is an issue faced by GWASs researchers when classifyingsamples as cases or controls."
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