path: root/gnqa/paper1_eval/src/data/datasets/old/experts_general1_dataset.json

{
  "question": [
    "How do I determine which gene in my QTL is causal for the trait?",
    "Why do males have two Y chromosomes and females only one?",
    "what type of dataset is useful for qtl mapping analysis in genenetwork2?",
    "What are the ethical considerations surrounding prenatal genetic testing and the selective termination of pregnancies based on genetic factors?",
    "What are the potential benefits and risks associated with gene editing technologies like CRISPR-Cas9?"
  ],
  "answer": [
    "To determine which gene in your QTL is causal for the trait, you can follow several steps. First, narrow down the QTL interval to a reasonable size. Then, sort through the genes within the interval to identify potential Quantitative Trait Genes (QTGs). This can be challenging as more than one gene may be involved and the function of some genes may be unknown. You can use techniques like LCMS to establish causal relationships between DNA loci and transcripts. Once potential QTGs are identified, you need to prove that a particular gene is involved in the quantitative trait under study. This can be done by complementation of a QTL, which can be achieved in several ways. You can also use mediation analysis to identify the causal gene between the genetic variant and the trait-of-interest. Finally, investigate whether the expression of these genes correlates with the phenotype(s) of interest. If the gene expression strongly correlates with the QTL phenotype, this further strengthens the assumption that this gene might be causal.",
    "Actually, males have one Y chromosome and one X chromosome, while females have two X chromosomes. This combination determines the sex of an individual.",
    "GeneNetwork utilizes datasets containing phenotypes and genotypes for QTL mapping analysis. These datasets may include genetic, genomic, and phenotypic data. The data are often formatted and normalized, and come with metadata. The database also includes data on complex phenotypes ranging from gene expression to behavior in standard inbred strains, and panels of mouse recombinant inbred strains. Rat and Arabidopsis populations are also represented in the database.",
    "The ethical considerations surrounding prenatal genetic testing and selective termination of pregnancies based on genetic factors include the potential for implicit pressure on individuals to violate personal ethics to reduce financial burden on society, the risk of routinization of testing leading to social or medical expectations of testing in all eligible individuals, and the potential compromise of values of informed consent and individual autonomy. There are also cultural and religious beliefs to consider, as well as the potential psychological impact on parents who may feel guilt if they are carriers of genetic conditions. Furthermore, the decision to terminate a pregnancy based on genetic factors is a joint decision between parents, and the involvement of extended family members in this process varies greatly across different cultures.",
    "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."
  ],
  "contexts": [
    [
      "Prior belief or knowledge about the number of true causal and true independent links that might be expected in a typical QTL, depending on the study design, should be considered to safeguard against high false-positive rates (low positive predictive values). In studies that involve mapping gene expression (eQTL), protein (pQTL) or metabolite (mQTL) traits, information about co-localization of QTL and genes that are functionally linked to the trait provides information about the likelihood of causal links.",
      "The next step is to investigate whether the expression of these genes correlates with the phenotype(s) of interest.This would suggest a chain of causality: a variant within a gene causes a change in its expression, and the expression of that gene correlates with expression of a phenotypic trait of interest.To do this, we created a correlation matrix between all genes within a QTL with a cis-eQTL in any brain tissue as well as the phenotypes that contributed to the QTL (Supplementary Table S6).Any gene with a cis-eQTL and a significantly correlated expression was considered a good candidate.If the gene only had a cis-eQTL and correlation in a single brain region, then it suggested that this brain region might also be of interest for the phenotype (adding another link to this chain).",
      "One possible approach to facilitate this endeavor is to identify quantitative trait loci (QTL) that contribute to the phenotype and consequently unravel the candidate genes within these loci. Each proposed candidate locus contains multiple genes and, therefore, further analysis is required to choose plausible candidate genes. One of such methods is to use comparative genomics in order to narrow down the QTL to a region containing only a few genes. We illustrate this strategy by applying it to genetic findings regarding physical activity (PA) in mice and human.",
      "Network analyses We now have two QTL, and we have picked potentially interesting genes within each, but now we want to build up more evidence for which gene in our QTL interval is causal. The first, and most obvious way, is to see what genes our trait of interest correlates with, in tissues that we expect to be related to the trait. We calculated the Spearman\u2019s correlation between the trait BXD_17850 and all probes with expression data in T helper cells (GN319).",
      "Another approach to help to determine if a gene located near the mapped QTL would have effects to influence the quantitative trait will be to use genetically engineered mice to determine if altering the expression of a candidate gene will alter the phenotype of interest (38). However, it is possible that a quantitative trait is a combined effect of multiple genes located near the QTL (39).",
      "With a known QTL and a body of evidence suggesting possible roles for the affected gene, phenotypes can be predicted that may be modulated as a result of this sequence variation. If this phenotype is of interest, it can be directly measured and a traditional \u2018forward\u2019 QTL analysis carried out to confirm the prediction. Such an approach is extremely attractive when the enormous cost and time required for phenotyping a large panel is considered.",
      "The first step is to narrow down the list of candidate causal genes within a Fig 1. Interval mapping of oviduct gross pathology across the BXD strains  Quantitative Trait Locus (QTL)\u2014a reveals a QTL on distal Chr 3. The L RS values are plotted in blue across the  chromosomal region containing genome and measure the strength of the association between  sequence variants strongly chromosome and Mb position (top and bottom X-\u00ad\u2010axis, respectively) and  associated with phenotypic phenotype expression. Allele contribution is shown by the red (C57BL/6J)  and green (DBA/2J) lines. Red and grey horizontal lines indicate genome-\u00ad\u2010 variation.",
      "A special case is the correlation of the target phenotype with the expression of the priorized gene(s) (RNA or protein amounts). This refers to colocalization of the QTL of the target phenotype with the eQTL position. Correlation can also be examined between the target QTL phenotype and expression of all genes in the QTL interval. If the gene expression strongly correlates with the QTL phenotype, this further strengthens the assumption that this gene might be causal (see Note 12). For performing a correlation analysis: \u2013  Go to the Trait Overview Page, as described in step 3, point 1.",
      "QTL mapping of traits in mouse cohorts often ends up with a genetic locus, composed of a list of candidate genes. Several studies proposed the use of mediation analysis to identify the causal gene (mediator) between the genetic variant (independent variable) and the trait-of-interest (dependent variable) (Figure 1.4B) [7, 47, 61, 77]. Mediation analysis can be used either on gene expression levels to identify the regulatory mechanisms [7, 47, 61], or on phenotypic traits to discover the potential causal drivers contributing to the phenotypic variances [77] (Figure 1.4C upper).",
      "1a). Second-generation offspring are then phenotyped and genotyped, and linkage analysis is carried out to identify a region that is associated with the trait1. This approach has led to the identification of thousands of quantitative trait loci (QTLs) for various phenotypes and diseases. However, each QTL region is large, often tens of megabases, and contains hundreds of genes. The process of identifying the causal variant and the gene involved is therefore difficult and costly. Of the thousands of QTLs identified, only a small fraction of genes has been identified. NIH-PA Author Manuscript  \u00a9 2012 Macmillan Publishers Limited.",
      "Network analyses We now have two QTL, and we have picked potentially interesting genes within each, but now we want to build up more evidence for which gene in our QTL interval is causal. The first, and most obvious way, is to see what genes our trait of interest correlates with, in tissues that we expect to be related to the trait. We calculated the Spearman\u2019s correlation between the trait BXD_17850 and all probes with expression data in T helper cells (GN319).",
      "10 JUNE 2016 \u2022 VOL 352 ISSUE 6291  aad0189-5 R ES E A RC H | R E S EA R C H A R T I C LE  Solving QTLs: Finding the quantitative trait gene For cis-QTLs, the causal factors can be quickly identified: With few exceptions, they will be driven by variants within the gene itself or immediately adjacent. For trans-QTLs, mQTLs, and cQTLs, the identification of the causal quantitative trait gene (QTG) is challenging due to the width of the QTLs.",
      "Once the QTL interval is reduced to a reasonable size, the next step in the process involves sorting through the genes within the interval and attempting to determine which is the QTG. This step is daunting because more than one gene may be involved and the function of some genes within the interval may be unknown. Until recently, this step emphasized the detection of polymorphisms within coding sequence (reviewed in Korstanje and Paigen, 2002 and Glazier et al. 2002); for a polymorphism that produces an amino acid substitution, one can often infer and then test for a functional consequence.",
      "To understand the genetic networks that underlie quantitative variation in the trait, it is also very important to discover genes whose expression is correlated with the trait after accounting for the known effects of the QTL on the trait. Many of these genes may have expression that is associated with QTL genotype, and would therefore be identified as important via the tests described above. Other  genes, however, may have expression values that are correlated with the trait but unassociated with genotype at the QTL.",
      "The approach is motivated by the fact that a research project is often focused on a specific classical quantitative trait. If a major QTL for this classical trait has been identified, it is often desirable to test whether this QTL is also associated with the transcription level of any genes, which will provide clues as to which genes belong to the pathway that the QTL uses to modulate the classical trait.",
      "Confirmation of Candidate Genes The next step is to prove that a particular gene is involved in the quantitative trait under study. This is done by complementation of a QTL, which can be achieved in several ways (9\u201311,40). In principle, transgenic complementation is the most straightforward. This approach has been used successfully to demonstrate that Pla2g2a was the correct candidate gene for Mom1, a modifier of the apcmin allele that causes adenomatous polyposis coli (41).",
      "So, how do you go about planning and performing a QTL study, and how do you identify the responsible gene within a QTL that you have identified? Generally, one starts by performing a strain survey to find two parental inbred strains that have a markedly different trait. One can now look up many different traits of inbred mice online at the Mouse Phenome Database (http://phenome. jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home). However, the trait you may want to study may not be present in wild type mice, so you may want to cross a mutant (or genetically engineered) strain onto several inbred strains.",
      "Along with correlations, this tool also derives new traits representing the principal components (Figure 2d). The user can add these principal components to their Trait Collection and proceed to perform QTL mapping, as in the case of a single trait QTL mapping. The R/QTL (Broman et al. 2003) and R/CAPE (Tyler et al. 2013) packages can be used for deeper analysis of epistasis and pleiotropy for multiple traits and multiple regulatory loci. Prioritizing Candidate Genes 7  Author Manuscript  Following the identification of a significant QTL, focus shifts to identifying the particular gene(s) that cause the QTL.",
      "The investigators first identified all QTLs associated with a classical phenotype and then winnowed the list of potentially associated gene-expression traits on the basis of their correlation or eQTL overlap with the phenotype of interest. Candidate genes then were ranked by applying  the LCMS technique, which uses the eQTL data to establish causal relationships between DNA loci and transcripts as well as between transcripts and phenotypes and finally identifies a model that best fits the data.",
      "The goal of QTL mapping is clearly the identification and eventual confirmation of candidate genes (QTGs) underlying the phenotype. The evidence required for such confirmation has engendered much discussion (Complex Trait Consortium 2003; Glazier et al. 2002) and is likely to vary depending on the nature of the trait and specific resources available to pin down underlying genes (e.g. availability of knock-in or knock-outs, specific antibodies, siRNA, etc.). The paucity of QTGs meeting such multifaceted standards is testament to the difficulty of narrowing the confidence interval sufficiently to identify and test suitable candidate genes (Flint et al."
    ],
    [
      "Y chromosome in peripheral blood cells increases with age in men (6) and is correlated with increased risk of cancer mortality and Alzheimer's disease (6,7).X chromosome mosaicism in women also increases with age (8), as does autosomal mosaicism in both sexes (9,10).Recent studies have shown that the prevalence of age-related mosaic abnormalities is greater in men than women (9,10); however, mechanisms underlying the sex differences observed in chromosomal mosaicism in humans are unknown.",
      "Recent reports suggested a role of Y chromosome loss in risk for all-cause mortality and common age-related disease such as cancer, Alzheimer disease as well as severe atherosclerosis [12][13][14][15][16][17][18][19][20].Building on such reports, we aimed to evaluate the contribution of male Y chromosome mosaicism to the risk for late-stage AMD.",
      "Box 1. Sex-specific cytonuclear interactions  Several predictions about the nature of cytonuclear conflicts follow from the patterns of chromosomal inheritance (Table I).In a mated pair of animals, mtDNA is co-transmitted with half of the autosomal genes, two-thirds of the X-linked genes and none of the Y-linked genes [76].This predicts that, relative to the autosomal case, positive nuclear-mitochondrial interactions are more likely to evolve for X-linked loci whereas deleterious interactions between Y-linked genes and mtDNA should accumulate (or cannot be purged efficiently).",
      "In addition to genetic data, the 9p Network Cohort dataset also lists the gender for all 719 individuals.Of these individuals, 406 individuals are female and 313 are male, indicating a female bias (Binomial test p \u00bc 0.0006).This result was surprising considering that no female bias has been previously reported in 9p deletion and duplication syndromes.A possible explanation for the significant bias in the 9p Network Cohort dataset is the XY sex reversal phenotype, which is commonly observed in individuals with 9p deletion syndrome.This phenotype could lead to individuals with XY sex chromosomes being listed in the dataset as having a female gender.To further examine this hypothesis, we subset our dataset to include only the 236 individuals whose sex chromosomes are listed in their genetic information.For this much smaller subset, 125 individuals had female sex chromosomes and 111 had male sex chromosomes, indicating no significant sex bias (Binomial test p \u00bc 0.4).We also found no significant gender bias in this group (Binomial test p \u00bc 0.2), although we did confirm that four of the individuals with XY sex chromosomes had a gender of female.This comparison suggests that the XY sex reversal phenotype may be responsible for a female gender bias, but not a sex bias, in 9p deletion and duplication syndrome cohorts.",
      "Duplicated variants with multiple alternative alleles and variants in sex chromosomes X and Y",
      "Autosome-One of the numbered, or nonsex, chromosomes (1 through 22).X and Y are the sex chromosomes.",
      "Given such a high abundance of young male-biased genes, we asked whether their parental genes are also male-biased.We found that fewer parental genes of X-linked male-biased duplicates were also male-biased (20%, 2/10) compared to the parental genes of autosomal young male-biased duplicates (32%, 12/37).These data, despite the small sample sizes and being statistically not significant, may suggest that compared to autosomal young genes, X-linked young genes more often evolved novel male-biased expression.However, as the majority of young genes are the result of intrachromosomal duplication events, the pattern might also reflect the fact that X-linked old genes are less likely to be male-biased.",
      "A slight excess of X-linked female-biased genes was also detected (Fig. 2).Although most of them are old, a few recently arose on the X chromosome over 4 to 6 Myr in the common ancestor of the D. melanogaster and D. simulans clade (branch 5).This can be interpreted in the context of the dominance model of the sexual antagonism hypothesis.In this case, a dominant, X-linked gene that is favorable to females but disadvantageous for males can become fixed.The slow accumulation of female-biased genes in the X reflects an overall low rate of female gene origination, either due to a small dominance effect (the degree of dominance h!1/2), or a minor disadvantageous effect on males (the ratio of fitness effects of male relative to female k!0) along with a favorable effect on females (Vicoso and Charlesworth 2006, Equation 10).",
      "Regarding the second step in the evolution of male-biased genes, namely X!A transposition, sexual antagonism favorable for autosomal fixation (Vicoso and Charlesworth 2006) and/or MSCI (Lifschytz and Lindsley 1972;Betran et al. 2002) may play a role in this process.On the other hand, the within-chromosomal duplication rate is higher than the between-chromosomal duplication rate (Emerson et al. 2008), which may contribute to the slow pace of X!A transposition.",
      "It has been observed that male-biased genes in Drosophila are overrepresented on autosomes (Parisi et al. 2003;Ranz et al. 2003).Consistent with this result, a dynamic process that can explain the nonrandom autosomal distribution has also been observed, in which autosomal new genes with X-linked parental genes are often male-biased.Specifically, a significant excess of autosomal testisexpressed retrogenes were identified as RNA-duplicates of X-linked parental genes (Betran et al. 2002).Recently, similar X!A gene traffic was observed in the DNA-level duplication and relocation data set of the Drosophila genus (Vibranovski et al. 2009b), and was further confirmed for DNA-level duplications in the D. pseudoobscura neo-X chromosome (Meisel et al. 2009).In addition, selective extinction of neo-X linked male-biased genes also occurred in D. pseudoobscura (Sturgill et al. 2007).These three lines of genome-wide investigation support a common pattern of outof-X traffic for male-biased genes, resulting in an enrichment of these genes on autosomes in the long term.",
      "It has been reported that the initial manifestations of new gene emergence, namely polymorphic duplicates, occur at a lower frequency on the X chromosome, thus indicating that these duplicates are subject to stronger purifying selection (Emerson et al. 2008).Therefore, the excessive fixation of X-linked duplicates might not occur via neutral processes.Positive selection could have facilitated the fixation of X-linked young genes in addition to driving their subsequent sequence evolution.",
      "Occasionally, Y chromosome DNA is detected in the maternal plasma, and the fetus appears to have female genitalia on sonographic examination.The underlying mechanisms for this include a twin demise, a maternal disorder of sexual differentiation, such as Swyer syndrome, or that the mother has undergone a bone marrow or solid organ transplant from a male donor (Bianchi, 2018;Hartwig, Ambye, Sorensen, & Jorgensen, 2017).",
      "Because of the differences in sex chromosome number, the sexunmatched comparison contains internal controls, i.e. , in this comparison, genes on the X-chromosome and Y-chromosome (but not those on the autosomes) should show copy number imbalances re\ufb02ective of a single copy change. We showed that the sample that is not sexmatched had readily detectable differences in aCGH signals for genes on the X and Y chromosomes. No such patterns were evident for the autosomes of the sex unmatched individuals or for the sex chromosomes of the sex matched samples.",
      "Sex chromosome:  The X or Y chromosome in human beings that determines the sex of an individual.Females have two X chromosomes in diploid cells; males have an X and a Y chromosome.The sex chromosomes comprise the 23rd chromosome pair in a karyotype.See also: autosome Sex-linked: Traits or diseases associated with the X or Y chromosome; generally seen in males.",
      "X chromosome: One of the two sex chromosomes, X and Y. See also: Y chromosome, sex chromosome Y chromosome: One of the two sex chromosomes, X and Y. See also; X chromosome, sex chromosome",
      "The male heterogamety (XY) is the most common reported system, but many species have female heterogamety (ZW), and more occasionally, multiple chromosome systems  (Almeida-Toledo and Foresti, 2001; Devlin and Nagahama, 2002; Penman and Piferrer, 2008). Given the low resolution of optical microscopy to differentiate sex chromosomes in \ufb01sh, researchers have looked for an alternative in the tenfold longer meiotic chromosomes to detect mispairing tracts at the synaptonemal complex as an indication of the sex differentiated region with variable success.",
      "The exclusive female constitution of gynogenetic genomes provides information on the SD system, especially in a XX/XY system, where all female progenies are expected. If ZZ/ZW is the underlying system, male offspring always will be present, but the interpretation is more complex and will depend on the distance of the SD region to centromere and on the viability of WW offspring (Devlin and Nagahama, 2002; Penman and Piferrer, 2008). Induced triploids, on the other hand, are constituted by the combination of two female and one male genomes (Piferrer et al.",
      "The existence of a maternally silenced X-linked imprinted locus playing a role in social cognition could explain why males (X m Y) are more vulnerable to disorders of social cognition such as autism spectrum disorders than are females (X m X p ).The absence of the expression of this gene would not lead to autism itself, but would eliminate a putative protective factor, making an individual more susceptible to the effects of other ASD-predisposing genetic mutations or environmental factors.",
      "When meiosis takes place, a pair of chromosomes may fail to separate properly, creating a sperm or egg that has either two copies or no copy of a specific chromosome.This is a sporadic event and it is called nondisjunction.Nondisjunction can lead to an extra chromosome, called trisomy, or a missing chromosome, called monosomy (GHR, 2008l).Down syndrome is an example of trisomy.Individuals who have Down syndrome have an extra chromosome number 21. Turner syndrome is an example of monosomy.Girls who have Turner syndrome have only one X chromosome.This causes them to have short stature and be infertile (NHGRI, 2008l).",
      "X-Linked Inheritance  X-linked genetic disorders (also called sex-linked) are caused by gene mutations on the X chromosome.Most often X-linked genetic disorders are seen in males.Males inherit the X chromosome from their mother and the Y chromosome from their father.Because males have only one X chromosome, if they inherit a gene mutation on the X chromosome from their mother, they will have the disorder.Examples of X-linked genetic disorders occurring in males include hemophilia and Duchenne muscular dystrophy (GHR, 2008o)."
    ],
    [
      "The project also provides online analysis tools to allow identification of correlations within its data set. GeneNetwork (http://www.genenetwork.org), encompassing WebQTL, is a database of genotypes and complex phenotypes ranging from gene expression to behaviour in standard inbred strains, and six panels of mouse recombinant inbred strains including the two largest sets (BXD and LXS) of approximately 80 strains each. Rat and Arabidopsis populations are also represented. Approximately 1500 phenotypes spanning the 25 year history of these strains are incorporated in this public resource, many of which were retrieved from the literature.",
      "GN spares the user most of these problem. Data are formatted and normalized, and usually come with good metadata (often in the form of links to more information). This greatly simplifies QTL and eQTL analysis, candidate gene discovery, coexpression analysis, and hypothesis testing [3, 10].",
      "Suitable for quantitative genetics (QTL mapping) and systems genetics, including correlation and network analysis to compare associations between tissues and between other rodent or human data sets  Description and usage  [32]  [31]  [30]  [11]  References  Many of the Data Sets are amenable to systems genetics mapping and other methods and are accessible at GeneNetwork. The Description and Usage column provides details about the data set and potential usage.",
      "Bioinformatics All of the genetic analyses were carried out in GeneNetwork, which is an open source bioinformatics resource for systems genetics that exists as both a repository for genetic, genomic and phenotypic data together with a suite of statistical programs for data analysis that includes mapping and evaluating QTLs, examining phenotype/genotype correlations and building interaction networks. QTL mapping The QTL mapping module of GeneNetwork was used to identify QTLs for hippocampal morphometry and radial maze trait data. This module enables interval mapping, composite interval mapping and a pairwise scan option to identify epistatic effects.",
      "There are four options for QTL mapping on the GeneNetwork website: interval mapping, marker regression analysis, composite interval mapping, and pairscan analysis. In this case, interval mapping was used to compute linkage maps for the entire genome. The log of odds (LOD) score was used to assert that a causal relation exists between a chromosomal location and a phenotypic variant, such as Gsto1 expression variation.",
      "Webqtl is an online database [110] of linked datasets, including genotype and expression data, covering multiple species including mouse, macaque monkey, rat, drosophila, arabidopsis, plants and humans [60]. While this tool cannot be used to calculate eQTLs, it can be used to find and visualize eQTLs in different species, strains and tissues. It can perform single- and multiple-interval QTL mapping of up to 100 selected traits. Users can also upload their own trait data for populations included in the database. It can also calculate and display trait-correlation matrices and network graphs (also for up to 100 traits).",
      "GN spares the user most of these problem. Data are formatted and normalized, and usually come with good metadata (often in the form of links to more information). This greatly simplifies QTL and eQTL analysis, candidate gene discovery, coexpression analysis, and hypothesis testing [3, 10].",
      "Suitable for quantitative genetics (QTL mapping) and systems genetics, including correlation and network analysis to compare associations between tissues and between other rodent or human data sets  Description and usage  [32]  [31]  [30]  [11]  References  Many of the Data Sets are amenable to systems genetics mapping and other methods and are accessible at GeneNetwork. The Description and Usage column provides details about the data set and potential usage.",
      "QTL MAPPING AND QTG DISCOVERY IN THE RCC A variety of statistical methods and tools have been developed for QTL mapping and implemented in free software for public use. These methods are well suited for simple backcross and F2 RCC populations. R/qtl9,39 was developed for identi\ufb01cation of QTLs and higher order modeling. Another Web-based tool, GeneNetwork or WebQTL (GeneNetwork.org),40 was developed for QTL mapping and to explore associations between variants, molecular traits (e.g. , gene expression), and higher order phenotypes (e.g. , behavior) and facilitate QTG identi\ufb01cation.",
      "This enables gene expression correlation and interval mapping, candidate gene searches and multitrait analyses. Each exported dataset was subject to an interval mapping analysis, which uses GeneNetwork\u2019s embedded MapManager software (Manly et al . 2001) to perform Haley\u2013Knott regression. Empirical P values were derived using 1000 permutations using the incorporated permutation feature of WebQTL. The peak of each statistically significant (P -value <0.05) or suggestive (P -value <0.63) (Lander & Kruglyak 1995) QTL was determined based on empirical P values (Doerge & Churchill 1996). A one-LOD drop-off was used to determine the QTL confidence interval about each peak.",
      "The peak linkage value and position was databased in GeneNetwork and users can rapidly retrieve and view these mapping results for any probe set. Any of the QTL maps can also be rapidly regenerated using the same Haley-Knott methods, again using functions imbedded in GeneNetwork. GeneNetwork also enable a search for epistatic interactions (pair scanning function) and composite interval mapping with control for a single marker. Data quality control  We used two simple but effective methods to confirm correct sample identification of all data entered into GeneNetwork.",
      "QTL analysis All QTL mapping for phenotypes was performed using the WebQTL software module of the  170  GeneNetwork (www.genenetwork.org) [34]. Interval mapping to evaluate potential QTLs was calculated from the likelihood ratio statistics (LRS) as the software\u2019s default measurement of the association between differences in traits and differences in particular genotype markers. Another common measure score, the log of the odds (LOD) ratio, can be converted from the LRS (LRS/4.61). Suggestive and significant LRS values were determined by applying 1000  175  permutations.",
      "Unlike interval-specific haplotype analysis, which is most useful for narrowing a QTL shared by multiple crosses, genome-wide haplotype analysis requires only phenotype information from many inbred strains and can effectively narrow a QTL identified in only one experimental cross [36]. After narrowing the QTL to an interval that is !5 Mb using these bioinformatics techniques or classical experimental methods, strain-specific sequence and gene expression comparisons are effective for focusing on a few strong candidate genes (Figure 7).",
      "We considered QTL intervals that achieved genome-wide significance for one phenotype, and genome-wide suggestive for others, as highest priority for candidate gene analysis. The January 2017 BXD genotype file was used4 . Updated linear mixed model mapping algorithms are now available on GeneNetwork 25 (Sloan et al. , 2016), that account for kinship among strains. These new algorithms include GEMMA (Zhou and Stephens, 2012), pyLMM6 (Sul et al. , 2016), and R/qtl27 .",
      "The peak linkage value and position was databased in GeneNetwork and users can rapidly retrieve and view these mapping results for any probe set. Any of the QTL maps can also be rapidly regenerated using the same Haley-Knott methods, again using functions imbedded in GeneNetwork. GeneNetwork also enable a search for epistatic interactions (pair scanning function) and composite interval mapping with control for a single marker. Data quality control  We used two simple but effective methods to confirm correct sample identification of all data entered into GeneNetwork.",
      "There are four options for QTL mapping on the GeneNetwork website: interval mapping, marker regression analysis, composite interval mapping, and pairscan analysis. In this case, interval mapping was used to compute linkage maps for the entire genome. The log of odds (LOD) score was used to assert that a causal relation exists between a chromosomal location and a phenotypic variant, such as Gsto1 expression variation.",
      "eQTL mapping  QTL mapping was performed with GeneNetwork, an online bioinformatics resource featuring tools for systems genetic and complex trait analysis [9, 35]. QTL mapping involves entering VMB and CP iron data (strain means and SEM) as quantitative traits; the software generates whole-genome interval maps for each trait. The interval maps graphically illustrate phenotype\u2013genotype associations as peaks (QTL) indicating the strength of association between genomic polymorphisms and the quantitative trait throughout the genome.",
      "Genetic Mapping In this study we utilize GeneNetwork, a database containing phenotypes and genotypes, and also serves as an analysis engine for quantitative trait locus (QTL) mapping, genetic correlations, and phenome-wide association studies (PheWAS) (Sloan et al. , 2016; Mulligan et al. , 2017; Watson and Ashbrook, 2020). QTL analysis involves connecting phenotype data with genotype data to examine genetic variation in traits controlled by multiple genes and their interaction with the environment (also called complex traits)(Lynch et al. , 1998; Myles and Wayne, 2008; Goddard et al. , 2016).",
      "Once the resulting record set of the query is returned, it can be further restricted by selecting relevant records based on attached annotations before forwarding it for further analysis. To map genetic loci associated with mRNA abundance or trait phenotypes, any one of the three QTL mapping functions currently employed by GeneNetwork's WebQTL module can be used. These are 1. interval mapping, 2. single-marker regression, or 3. composite mapping [29,30].",
      "genenetwork.org/) a set of 3795 markers. Linkage is reported with genome-wide significance levels based on 2000 permutation tests. Two types of QTL mapping analyses\u2013simple mapping using the Haley\u2013 Knott regression equation, and composite interval mapping\u2013were utilized in this study. Simple interval mapping was performed to illustrate the significance of any QTLs that regulate the TID. As a secondary analysis, composite interval mapping which controlled for the influence of Tyrp1 was also performed with the goal of identifying any secondary QTLs that may have been masked by the major QTL on Chr 4."
    ],
    [
      "A number of additional ethical implications must be considered.Associating financial investments with the prevention of disease, especially where reproductive decisions are involved, requires sensitivity, caution, and ethical rigor.Funding decisions based on imputed cost-savings must not result in implicit pressure on individuals to violate personal ethics to reduce financial burden on society.As discussions regarding prenatal testing have demonstrated, 35 is risk that \"routinization\" of testing may lead to social or medical expectations of testing in all eligible individuals.These expectations, if linked with financial incentives for the health system, could risk applying implicit pressure on serious, and potentially irreversible, personal decisions.Such expectations, if applied at the population level, could risk becoming normalized, compromising the values of informed consent and individual autonomy.",
      "With regard to pregnancies affected by a genetic condition identified through population carrier screening, we modeled the decision to terminate affected pregnancies conservatively (0.50).This is despite the literature suggesting rates above 0.90 for elective TOP for conditions such as Down syndrome 33 and SMA. 34We recognize this issue is controversial, and that laws and ethical positions vary considerably between countries/ jurisdictions.Variations in population attitudes based on age, religion, and other factors, as well as the criticality of preserving individual choice, were acknowledged in adopting this highly conservative estimate.",
      "The use of genetic testing from pre-conception through adulthood is expanding rapidly.As a result of this expansion, new ethical issues are emerging related to genetic testing and informed consent.These new issues create ethical challenges for nurses and all healthcare providers.Currently expanding areas include newborn screening and genetic testing of children.These new ethical challenges will be described below.",
      "The use of genetic testing from pre-conception through adulthood is expanding rapidly. Psychological risks for parents who are carriers may include parental guilt.",
      "Ethnic and cultural backgrounds may also play a role in the decisions that families make regarding prenatal testing.Moyer et al. (1999) concluded that Caucasian women more often undergo prenatal diagnoses than African American or Asian women, or Latinas.Furthermore, Awwad et al. (2008) found American couples less inclined to involve extended relatives in the prenatal decision-making process than Native Palestinian couples.Both of these examples clearly indicate that cultural differences can impact the ways in which families negotiate prenatal decisions.Further research needs to investigate how different families engage in such discussions and decision-making processes, especially as prenatal testing becomes more common and better able to predict or prevent a wider range of genetic conditions.Tightly closed ethnic groups remain at high risk of serving as carriers for genetic mutations, but the management of this possibility varies greatly.For example, some Ashkenazi Jewish groups use screening for mutations for Tay-Sachs disease (TSD) as the basis for rabbinical marriage advice; whereas, children born to Amish families in Pennsylvania more often present with glutaric aciduria type 1 (GA1) but, given their beliefs, parents tend not to accept prenatal testing because of the implication of abortion (McKusick, 2000).",
      "Researchers studying factors that contribute toward a couple's choice to undergo prenatal testing have determined that partners base their decision upon several factors, including, but not limited to: parental beliefs about abortion, attitudes regarding disability and their \"perceptions of the usefulness of having the information revealed by genetic tests\" (Moyer et al., 1999, p. 522).Abortion beliefs constitute a key issue in the decision-making process.Even though a majority of parents receiving abnormal prenatal test results terminate their pregnancies (Redlinger-Grosse, Bernhardt, Berg, Muenke, & Biesecker, 2002), Moyer et al. noted that, when asked, more families reported that they would make use of prenatal testing than would be willing to terminate a pregnancy.The decision to continue or terminate a pregnancy after prenatal testing Downloaded by [University of the Sunshine Coast] at 10:32 05 August 2017 comprises a joint decision between both parents (e.g., Awwad et al., 2008;Beeson & Golbus, 1985); however, the nature of the conversations leading to the decision and the involvement of extended family members in the decisionmaking process remains highly understudied.",
      "The Genetic Divide(s) and Communication  The ability of scientists to \"map\" disease through several generations (Collins, 1999) raises practical and ethical issues of access to resulting opportunities and creates family communication challenges.Currently, prenatal testing for chromosomal diseases has become increasingly common (Moyer et al., 1999).Options such as pre-implantation genetic diagnosis (PGD) can identify over 1,250 disease-related mutations creating an opportunity for parents to select unaffected embryos for implantation in the womb (R. M. Green, 2008).Test results provide potential parents with information that may lead to decisions involving intervention in the genetic makeup of future children.Although some families welcome such options, others may be unable or unwilling to consider such procedures, due to fi nancial concerns or moral/ethical/religious beliefs.",
      "Privacy Issues  Finally, privacy issues should be seriously considered when the use of genetic testing is contemplated, especially with respect to whole-genome sequencing of healthy people.It is an unanswered question under what circumstances, to what extent, and by what means genetic data should be incorporated into the medical record.Although easy access to such data could be helpful to providers in improving patient care, it remains to be seen how other parties (eg, insurance companies) might act on the data in ways that do not benefit patients.The US Congress acted to prohibit discrimination by employers and health insurers on the basis of genetic testing with the Genetic Information Nondiscrimination Act in 2008, but further safeguards will undoubtedly be needed as the health implications of genetic data become clearer.",
      "The ethical evaluation of genetic testing in children is traditionally based on the balance of clinical benefits and risks (American Society of Human Genetics Board of Directors and the American College of Medical Genetics All correspondence concerning this article should be addressed to Benjamin Wilfond, MD, Treuman Katz Center for Pediatric Bioethics, Seattle Children's Hospital, Metropolitan Park West M/S: MPW 8-2, 1100 Olive Way, Room 876, Seattle WA 98101, USA.E-mail: benjamin.wilfond@seattlechildrens.org Board of Directors, 1995;Andrews, Fullerton, Holtzman, & Motolsky, 1994;Clarke, 1994;Wertz, Fanos, & Reilly, 1994).In the early 1990s, when there were only scant data about children who had received genetic tests results, the presumption was to give greater weight to the potential risks and to restrict testing.However, this criterion is not necessarily consistent with the general practice of respecting broad parental discretion in health care decisionmaking for and on behalf of their children.In general, parents are the presumed decision makers for their children and their decisions are respected unless they are abusive or neglectful (Buchanan & Brock, 1989;Goldstein, Freud, & Solnit, 1979;Ross, 1998).The tension between assessments of benefits and risks made by health care providers and policy makers, and the procedural respect owed to parental authority will be clearly tested as the ability to conduct and interpret whole-genome sequencing and related technologies gain in momentum.",
      "Ethical Considerations in Developing Policy for ''Comprehensive'' Genomic Testing  In the near future, genomic testing is likely to become more accessible and will provide both information about the risks of common conditions such as heart disease, diabetes, and hypertension as well as predictions about individual responses to specific pharmaceuticals and other medical therapies (Aspinall & Hamermesh, 2007).Over time, the number and range of conditions for which such testing is available is likely to expand to include more behavioral traits, ranging from information about anxiety and depression, to attention and addiction (Rothstein, 2005).",
      "Objective Ethical evaluation of genetic testing in children is traditionally based on balancing clinical benefits and risks.However, this focus can be inconsistent with the general practice of respecting parental decision-making about their children's health care.We argue that respect for parental decision-making should play a larger role in shaping pediatric genetic testing practices, and play a similar role regarding decisions to use emerging genomic technologies.Methods Genomic testing involves the examination of thousands of DNA markers spanning genes throughout the genome and their interrelationships, yielding virtually limitless interpretations.We presume that parents and providers should proceed cautiously in applying genomic testing in children, as we explore how genomic testing will stress the fault lines of the traditional ethical analysis.Results Empirical data about the psychosocial risks and benefits of genetic testing of children do not reveal serious harms, yet virtually no such data exist yet about genomic testing.Unless empirical social and behavioral data indicate that genomic testing is highly likely to cause serious harms to the children, parental decisions to obtain comprehensive genomic testing in their children should be respected.Once comprehensive genomic testing of children becomes routine, resultant information may be more easily integrated by families than anticipated.Conclusions Research on the social and behavioral impact of comprehensive genomic testing on children and their families is needed to further inform parents, clinicians, and policy makers.",
      "Objective Ethical evaluation of genetic testing in children is traditionally based on balancing clinical benefits and risks.However, this focus can be inconsistent with the general practice of respecting parental decision-making about their children's health care.We argue that respect for parental decision-making should play a larger role in shaping pediatric genetic testing practices, and play a similar role regarding decisions to use emerging genomic technologies.Methods Genomic testing involves the examination of thousands of DNA markers spanning genes throughout the genome and their interrelationships, yielding virtually limitless interpretations.We presume that parents and providers should proceed cautiously in applying genomic testing in children, as we explore how genomic testing will stress the fault lines of the traditional ethical analysis.Results Empirical data about the psychosocial risks and benefits of genetic testing of children do not reveal serious harms, yet virtually no such data exist yet about genomic testing.Unless empirical social and behavioral data indicate that genomic testing is highly likely to cause serious harms to the children, parental decisions to obtain comprehensive genomic testing in their children should be respected.Once comprehensive genomic testing of children becomes routine, resultant information may be more easily integrated by families than anticipated.Conclusions Research on the social and behavioral impact of comprehensive genomic testing on children and their families is needed to further inform parents, clinicians, and policy makers.",
      "To the extent that ''personal meaning'' gains wider acceptance as a legitimate criterion for expanding the availability of new tests and applications of genomic technology, the current policies and practices of restricting some genetic testing of children and mandating other tests will need to be reevaluated.There will be some parents who will find the information that becomes available through new technologies and data useful in shaping their parenting practices, while others will be more skeptical of their value.These disparate parental judgments may be independent of professional assessments of clinical validity and utility.Extrapolating from the empirical data about predictive genetic testing of children in at-risk families discussed earlier, we speculate that once comprehensive genomic testing of children becomes routine, the information may be more easily integrated by families than might be predicted.This is not meant to imply that whatever information parents want about their children should be provided carte blanche.Clearly, education and counseling will be crucial to ensure that families understand the limitations of the information.However, restrictions and mandates should be based on a criterion of risk of serious harm (Diekema, 2004).Given the lack of data confirming harm and the related data that indicate children may fare better than anticipated, such restrictions and mandates cannot be justified.Policies and practices will also need to clarify the role of the older adolescent in the decision-making process, although the issues related to balancing and assessing parental and adolescent interests and preferences goes beyond the focus of this article.This is also not meant to ignore the professional and moral obligation to educate parents and to help parents make good decisions on behalf of their children.It is morally appropriate for providers to strongly recommend particular tests in infancy and young childhood (i.e., PKU testing), and to strongly discourage other tests (e.g., ApoE testing of children for adult onset Alzheimer disease and heart disease because ApoE is not predictive but only provides an increased relative risk and has limited sensitivity and specificity) (Roberts, Cupples, Relkin, Whitehouse, & Green, 2005).Selective and directive recommendations are a routine aspect of pediatric practice.However, it will become increasingly important for professional organizations to begin to reconcile their support for mandatory genetic testing for some conditions and their support for restrictions for other conditions with the broad discretion that parents have and need in the health care arena in order to promote their children's well-being.",
      "What limits should be imposed, if any, need to be determined prior to commercial feasibility.In this article, we consider how genetic testing decisions for children have been made traditionally and how the anticipation of comprehensive genomic testing in the near future will stress the fault lines of traditional approaches.The potential for comprehensive genomic testing in children could shift the equilibrium towards expanding or reducing parental discretion, and forces us to reexamine the evidence for our genetic testing policies and practices.We will highlight specific domains where further empirical social and behavioral research is necessary to inform policy and practice.",
      "Prenatal genetics is largely practiced by maternal-fetal medicine specialists due to severe deficiency in the number of qualified clinical geneticists.Recent years have witnessed a tremendous growth in the demand for chorionic villous sampling and amniocentesis for the diagnosis of single gene disorders.At KFSHRC alone, the number of prenatal samples that are tested for single gene disorders has increased from 5 in 2004 to 250 in 2013.Therapeutic abortion is permitted by law if performed within 120 days from the time of fertilization in order to comply with the Islamic view of the timing of ensoulment (Alkuraya and Kilani 2001).However, the approved indication for the procedure, which is \"severe malformation\", must be authorized by three attending-level physicians.The definition of \"severe\" is left to the discretion of the medical team after consulting with the family.For example, intellectual disability is a common indication for many therapeutic abortion procedures.Contrary to commonly held views, we have shown that early prenatal diagnosis is the method of choice for couples who had one or more children with single gene disorders, as long as they are provided with a culturally sensitive genetic counseling that addresses their religious and cultural concerns (Alkuraya and Kilani 2001).Nearly 45% of these couples opt for early prenatal diagnosis compared to 35% who choose preimplantation genetic diagnosis (PGD) (Alkuraya 2013a).PGD is available freely at KFSHRC but is also provided by the private sector.Noninvasive prenatal screening using cell-free fetal DNA in maternal blood is quickly becoming integrated in prenatal care.KFSHRC offers this test routinely to all pregnant women regardless of their perceived risk and the MOH is considering making this test available throughout its vast network of hospitals and medical centers.",
      "Social and psychological implications of accessing genetic services and information.",
      "A corollary of the predictive power of genetic information is the limited ability to prevent or treat many conditions with significant genetic factors involved.Indeed, virtually all of the complex ethical and legal issues relevant to genetic testing would disappear if there were effective preventions or treatments available for genetic conditions.The ability to predict future disease in conjunction with a limited ability to do much about it has important social and psychological implications that must be addressed in conducting genetic research.",
      "Interpretations of the literature will likely mirror the priorities and evaluative tendencies of the reader.Are you willing to accept the overall trends in genetic and genomic testing evaluation and to trust that the existing clinical approaches will apply informed consent appropriately while identifying and supporting the rare individual who has a serious adverse response to the testing?If so, you might advocate that attention be turned more toward other issues relevant to the effective implementation of genetic and genomic testing.Or do you feel a strong need to understand in more detail the possible psychosocial harms of the testing, particularly the subtler impacts or responses of individuals who do not fit the norm?In that case, you would likely encourage renewed and innovative efforts to study the psychosocial consequences of the receipt of risk information from genetic and genomic testing.",
      "Other social issues require our attention if genomic medicine is to benefit our patients.How should genetic tests be regulated?What, if any, are the appropriate uses of direct-to-consumer marketing of genetic tests?The Internet has recently had a proliferation of genetic-testing sites that feature claims grounded in greed and pseudoscience, rather than in data or reality.How will health care providers and the public distinguish between these and responsible testing services, whether they are available through the Internet or in the hospital?",
      "Environmental Factors  As widespread use of genetic testing increases, it is the responsibility of the medical community to ensure its equitable use across socioeconomic and cultural spectrums."
    ],
    [
      "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 Applications  Despite 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.",
      "Conclusions  There 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 tools  Off-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 technologies  The 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-Cas9  CRISPR 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.",
      "INTRODUCTION  Genome 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 improvement  Reports 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)."
    ]
  ]
}