Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality.

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.

BackgroundGene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™).ResultsWe present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum.ConclusionsUsing an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.

Pichia pastoris has gained much attention as a popular microbial cell factory for the production of recombinant proteins and high-value chemicals from laboratory to industrial scale. However, the lack of convenient and efficient genome engineering tools has impeded further applications of Pichia pastoris towards metabolic engineering and synthetic biology. Here, we report a CRISPR-based toolbox for gene editing and transcriptional regulation in P. pastoris. Based on the previous attempts in P. pastoris, we constructed a CRISPR/Cas9 system for gene editing using the RNA Pol-III-driven expression of sgRNA. The system was used to rapidly recycle the selectable marker with an eliminable episomal plasmid and achieved up to 100% knockout efficiency. Via dCas9 fused with transcriptional repressor (Mix1/RD1152) or activator (VPR), a flexible toolbox for regulation of gene expression was developed. The reporter gene eGFP driven by yeast pGAP or pCYC1 promoter showed strong inhibition (above 70%) and up to ~ 3.5-fold activation. To implement the combinatorial genetic engineering strategy, the CRISPR system contained a single Cas9-VPR protein, and engineered gRNA was introduced in P. pastoris for simultaneous gene activation, repression, and editing (CRISPR-ARE). We demonstrated that CRISPR-ARE was highly efficient for eGFP activation, mCherry repression, and ADE2 disruption, individually or in a combinatorial manner with a stable expression of multiplex sgRNAs. The simple and multifunctional toolkit demonstrated in this study will accelerate the application of P. pastoris in metabolic engineering and synthetic biology. KEY POINTS: • An eliminable CRISPR/Cas9 system yielded a highly efficient knockout of genes. • Simplified CRISPR/dCas9-based tools enabled transcriptional regulation of targeted genes. • CRISPR-ARE system achieved simultaneous gene activation, repression, and editing in P. pastoris.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

Molecular characterization of the gene feminizer in the stingless bee Melipona interrupta (Hymenoptera: Apidae) reveals association to sex and caste development

CRISPR/Cas9 system is a promising approach for gene editing in gene therapy. Effective gene editing requires safe and efficient delivery of CRISPR/Cas9 system in target cells. Several new multifunctional pH-sensitive amino lipids were designed and synthesized with modification of the amino head groups for intracellular delivery of CRISPR/Cas9 system. These multifunctional pH-sensitive amino lipids exhibited structurally dependent formulation of stable nanoparticles with the DNA plasmids of CRISPR/Cas9 system with the sizes ranging from 100 to 200 nm. The amino lipid plasmid DNA nanoparticles showed pH-sensitive hemolysis with minimal hemolytic activity at pH 7.4 and increased hemolysis at acidic pH (pH = 5.5, 6.5). The nanoparticles exhibited low cytotoxicity at an N/P ratio of 10. Expression of both Cas9 and sgRNA of the CRISPR/Cas9 system was in the range from 4.4% to 33%, dependent on the lipid structure in NIH3T3-GFP cells. The amino lipids that formed stable nanoparticles with high expression of both Cas9 and sgRNA mediated high gene editing efficiency. ECO and iECO mediated more efficient gene editing than other tested lipids. ECO mediated up to 50% GFP suppression based on observations with confocal microscopy and nearly 80% reduction of GFP mRNA based on RT-PCR measurement in NIH3T3-GFP cells. The multifunctional pH-sensitive amino lipids have the potential for efficient intracellular delivery of CRISPR/Cas9 for effective gene editing.

fshr: a fish sex-determining locus shows variable incomplete penetrance across flathead grey mullet populations.

Preface: development and evaluation of improved strains of insect pests for sterile insect technique (SIT) applications.

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, more nucleotides will increase the difficulties in plasmid construction and the risk of wrong integration in genome such as base-pair or fragment missing ( Gheysen et al., 1990 ). And also, in many organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly ( Sun et al., 2015 ). Thus, we have developed a method using single transcriptional unit (STU) CRISPR-Cas9 system to drive the expression of both Cas9 and sgRNAs from a single RNA polymerase II promoter to achieve effective genome editing in plants.

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas–mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3′ end of the target site abolished the cleavage activity completely. The mismatches at the 5′ end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3′ end. This approach provides a powerful method for genome engineering in plants.

The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene(PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.

A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A β-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of β-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.

Diamondback moth, Plutella xylostella (L.), is a specialist pest on cruciferous crops of economic importance. The large-scale use of chemical insecticides for the control of this insect pest has caused a number of challenges to agro-ecosystems. With the advent of the omics era, genetic pest management strategies are becoming increasingly feasible and show a powerful potential for pest control. Here, we review strategies for using transgenic plants and sterile insect techniques for genetic pest management and introduce the major advances in the control of P. xylostella using a female-specific RIDL (release of insects carrying a dominant lethal gene) strategy. Further, the advantages of gene drive developed in combination with sex determination and CRISPR/Cas9 systems are addressed, and the corresponding prospects and implementation issues are discussed. It is predictable that under the policy and regulation of professional committees, the genetic pest control strategy, especially for gene drive, will open a new avenue to sustainable pest management not only for P. xylostella but also for other insect pests.

Dynamics of sexual development in teleosts with a note on Mugilcephalus

RATIONALE Sophisticated computer software technology has recently been developed to provide data from the human genome project as well as from microarray and proteonomic studies. Initial studies using this technology have demonstrated the potential for major advances in several areas of biomedical research. Deoxyribonucleic acid (DNA) microarray techniques may be used to characterize profiles of changes in gene expression in unprecedented detail. For example, there have been studies of changes in the expression of thousands of genes in specific cells or tissues in response to aging, tissue injury, caloric restriction, drug treatment or alteration of a specific gene (e.g., mutation, and expression of an oncogenic transcription factor). Novel classification systems for leukemias, which have been generated by gene expression profiles, can potentially lead to optimal, targeted treatments. Gene expression profiles also have been used to characterize pathogenic factors in microorganisms. Together with newly developed laser microdissection techniques, which permit isolation of specific structures and even cell types from a tissue specimen, microarray techniques are beginning to be used for characterization of gene expression profiles within different components of tumors or inflamed tissues. DNA microarray techniques also may be used for large-scale studies of sequence variation. This type of application has the potential to revolutionize genetic mapping and, in large population studies, to identify genetic alterations that determine disease susceptibility. In addition, DNA microarray techniques may be used to characterize interactions between gene products. For example, microarrays have been applied to screens designed to identify inhibitors of specific biological response pathways. This type of application will be used in the future to identify substrates of enzymes, substrates for chaperones and ligands for receptors. It may be especially useful for identification of antisense oligonucleotides or antisense ribozymes for pharmacologic interventions. Finally, DNA microarray techniques have been used to screen for the function of products of disrupted genes in model systems. This type of application will be particularly important for understanding disease pathogenesis and for drug discovery projects. Proteonomics, or the analysis of complete sets of gene products at the protein level, has not been used widely, but these techniques may prove even more powerful for the identification of interactions between gene products. Thus, they may be designed for identification of substrates, inhibitors, ligands, agonists, antagonists, transcriptional activators and repressors. Application of these technologies to studies of the molecular basis of gastrointestinal (GI) health and disease in children may produce major advances relatively rapidly. The technology can be applied to almost all disorders affecting the GI tract, liver, biliary tract and pancreas. It can potentially facilitate the elucidation of genes altered in congenital anomalies or in inherited disorders that are monogenic, as well as the elucidation of genes that modify the clinical phenotype of monogenic disorders. Genetic traits that contribute to the development of polygenic diseases or determine susceptibility to infections, inflammatory diseases, toxins, drug reactions and developmentally determined or physiologic stressors also may be more rapidly identified with these technological advances. Application of these technologies may be particularly useful in understanding host responses to disease and injury, including intestinal adaptation, liver regeneration, responses to intestinal or biliary obstruction, host inflammatory response and host stress response. Indeed, these responses are thought to be critical determinants of the clinical phenotype and severity of many GI disorders. AREAS OF EMPHASIS Elucidate the Genetic Basis of Single-Gene Disorders and Genetic Determinants of Polygenic Diseases Research Goals Here we need to identify the genes altered in single-gene disorders, such as microvillus inclusion disease, neonatal iron storage disease, Alpers' disease and Shwachman syndrome. We also need to identify genes altered in polygenic diseases, such as inflammatory bowel disease and gluten-sensitive enteropathy. Finally, the genetic disorders that account for some general syndromes, such as intractable diarrhea of infancy and idiopathic neonatal hepatitis, need to be elucidated. Research Strategies This goal will require a combination of genetic mapping, microarray analysis, proteonomic analysis, and classical biochemical studies. A combined strategy was recently used to identify and characterize the ABC1 lipid transporter that is mutated in Tangier disease. Projected Timetable and Funding Requirements This area will be optimally addressed by investigator-initiated grants (e.g., RO1 grants). Center grants and program projects also will be effective strategies, particularly because they could permit the establishment of core facilities for computer software and microarray and proteonomic analysis. The initiative should be started immediately to take advantage of the new data being generated by the human genome project. Analyze Genotype-Phenotype Relationships and Genetic Modifiers of Single-Gene Disorders Research Goals Here we need to identify the genetic traits that determine the clinical phenotype of disorders in which the primary genetic abnormalities have already been identified. Cystic fibrosis and α1-antitrypsin deficiency are examples of monogenic disorders in which there is wide variation in phenotypic expression of target organ injury. Although rare, hereditary hemorrhagic telangiectasia is an example of a disorder with a striking genetically determined phenotypic variation. It is also very important to learn how childhood GI, hepatobiliary and pancreatic diseases can lead to carcinoma during the adult years. Examples include hereditary polyposis syndromes, inflammatory bowel disease and metabolic liver disease. Detailed information on tumorigenesis and cell survival (i.e., apoptosis pathways) is important for our understanding of the pathobiology of these diseases. This information also is important for the development and use of novel therapeutic strategies. For example, in the case of metabolic liver disease, transplanted hepatocytes have a selective advantage for growth and proliferation in the liver of the c14CoS albino mouse (a murine model of hereditary tyrosinemia) and will replace most of the damaged liver. However, it is not known whether a small number of residual dysplastic liver cells will become malignant and thereby limit the potential success of this type of novel therapy. Proteonomic techniques may be particularly useful for studying how GI diseases can lead to carcinoma. For example, proteonomic analysis was recently used to identify the expression of 43,302 proteins in normal human breast cells in anticipation of a comparison with protein expression in cells from breast cancer specimens. Research Strategies This goal will require a focus on basic biologic and pathobiologic work and should utilize genetic mapping, microarray and proteonomic techniques together with classical biochemical and cell biological studies. It will also require large-scale multicenter collaborative studies and registries to carefully characterize patients from a clinical perspective and provide material from these patients for genotyping. Projected Timetable and Funding Requirements This research should start immediately and will depend on investigator-initiated grants, program projects and center grants. The development of a registry for specific single-gene disorders, as defined by the other task force subcommittees, is strongly recommended. Funding should be solicited from several institutes at the National Institutes of Health (NIH) as well as from private foundations. A contract mechanism could work particularly well for the registries. Develop New Animal Models Research Goals It is recommended that the most sophisticated genetic engineering strategies be used to generate new animal models of GI disease. These strategies include transgenesis, targeted gene disruption, targeting mutagenesis, and conditional and inducible expression systems. It will be very important to ascertain the effect of genetic background in each animal model so that genetic modifiers of disease phenotype can be identified. Use of inducible or conditional expression systems will permit examination of the effects of developmental stage on disease phenotype. These studies are likely to be particularly informative for animal models of diseases that are affected by development. For example, in α1-antitrypsin deficiency, there is often clinical evidence of liver injury early in infancy and thereafter most patients enter a period in which there is marked lessening of liver injury. In some of these patients, liver disease recurs during adolescence. Some α1-antitrypsin-deficient individuals develop liver disease with or without hepatocellular carcinoma later during adult life. An animal model in which the hepatotoxic condition, retention of the mutant ZZ α1-antitrypsin molecule in the endoplasmic reticula of liver cells, is induced or abrogated at specific times during development may provide critical information about the mechanism underlying the effects of this disorder on the liver at different stages of development. Use of tissue-specific promoters will permit examination of the role of specific tissues in determining disease phenotype. Research Strategies This goal will require a collaborative effort between investigators, genetically altered mouse core facilities and their personnel, anatomists, pathologists, embryologists and physiologists. Characterization of the animal models will require microarrays and proteonomics. In fact, it is likely that at some point in the future, companies will be making the genetically altered mice and supplying them to investigators for this type of characterization. The development of a database for accessing information on all animal models is strongly recommended. Projected Timetable and Funding Requirements Program projects and center grants with core facilities would greatly facilitate this type of work. Consideration should be given to encouraging and facilitating collaboration between industry and academic research programs. Develop Novel Prophylactic/Therapeutic Interventions for GI Disease Research Goals It is recommended that proteonomic analytical systems be used to identify novel agonists and antagonists. In addition, three novel strategies for the prevention and treatment of genetically determined conditions should be studied in detail: Cell transplantation. Recent studies have shown that normal adult hepatocytes can replicate and replace much of the liver parenchyma, but only when transplanted into the background of an injured liver—specifically a liver injured by a metabolic defect, hereditary tyrosinemia. This form of therapy is therefore applicable to liver diseases in which the defect is cell-autonomous (e.g., many of the childhood metabolic liver diseases). In fact, recent studies have shown that hepatocyte transplantation may be effective in the treatment of type I Crigler-Najjar syndrome. There is reason to believe that cell transplantation strategies also can be used for injuries in other organs. Such studies will need to address the changes that occur in the diseased tissues, whether it is regenerative signals that permit cell replication, and how the cells that are to be transplanted can be optimally manipulated ex vivo (or in vivo after transplantation) using state-of-the-art genetic and molecular techniques. Chemical chaperones. The use of chemical chaperones for chemoprophylaxis of metabolic liver disease also is deserving of more detailed study. This class of compounds, which includes glycerol, trimethylamine oxide, deuterated water and 4-phenylbutyric acid (PBA), has shown to reverse the cellular mislocalization or misfolding of mutant membrane and lysosomal, nuclear, cytoplasmic and secretory proteins, including mutant CFTRΔF508 and ZZ α1-antitrypsin. In fact, PBA has been shown to have positive biochemical effects in an animal model of α1 -antitrypsin deficiency and in humans with cystic fibrosis. Recent studies have also suggested that competitive antagonists may have chaperone effects on mutant enzymes. One drug in this class, 1-deoxy-galactonojirimycin, a competitive antagonist of the lysosomal enzyme galactosidase A, mediates partial correction of the defect in localization of this enzyme in one type of Fabry disease. Recent studies have also shown that imino sugar compounds, which inhibit oligosaccharide side-chain trimming of glycoproteins, can partially reverse the mislocalization of mutant proteins such as ZZ α1-antitrypsin. Taken together, these compounds may have broad applicability in a variety of metabolic and genetic diseases of the liver, biliary tract, pancreas and GI tract, including α1-antitrypsin deficiency, cystic fibrosis, Wilson's disease, hemochromatosis, Gaucher's disease, Niemann-Pick disease and carbohydrate-deficient glycoprotein syndrome. Chimeric oligonucleotides. The use of chimeric ribonucleic acid (RNA)/DNA oligonucleotides for the prevention of tissue injury in genetic diseases is deserving of further investigation. Recent studies have shown that chimeric RNA/DNA oligonucleotides, based on the sequence of coagulation factor IX complex with lactose so that it could be taken up by asialoglycoprotein receptor-mediated endocytosis, are delivered to hepatocytes with high efficiency after intravenous administration. Moreover, the oligonucleotide complexes mended the mutation in more than 90% of the liver parenchymal cells in an animal model of factor IX deficiency. Studies are strongly recommended of genetic polymorphisms in drug metabolism, drug disposition, drug transporters and drug targets. Research Strategies This goal will require not only continued innovative cell transplantation gene transfer and pharmacology research, but also continued basic research. The basic research will focus on the developmental biology of the GI tract and liver, the biology of tissue response to injury and the biology of tissue regeneration. Presumably, the success of cell transplantation in treating genetic disease will depend on the development and differentiation of the cells to be transplanted, the ability to manipulate them ex vivo (and presumably in vivo after transplantation) using gene transduction techniques, the signals being generated by the response of the affected tissue to injury, and the need for the affected tissues to regenerate. Moreover, one would suspect that the response to injury and regeneration in the native tissue would depend on the developmental stage of the organ. Proteonomic techniques will be particularly useful for the identification of novel agonists and antagonists that bind to key cellular target molecules. This goal will also require large-scale, high-throughput screening methods to identify candidate drugs and genomic screens for the identification of polymorphisms in drug-metabolizing enzymes, transporters and targets. Projected Timetable and Funding Requirements This goal will require intensive partnerships with industry; in particular large pharmaceutical companies. Study the Basic Biology of Intestinal, Hepatic and Pancreatic Development and Responses to Infection, Inflammation, Tissue Injury and Other Physiologic/Environmental Stressors (e.g., Intestinal Adaptation and Liver Regeneration) Research Goals An enhanced understanding is required of the molecules involved in development and differentiation of the GI, hepatobiliary and pancreatic systems. In addition, how these tissues specifically respond to conditions associated with the expression of clinical disease is required. The intestinal adaptation response and liver regenerative response to injury are examples of signal transduction systems that are fundamental to determining whether a host will develop clinical disease and how severely the subject will be affected. Research Strategies Microarray and proteonomic techniques are particularly well suited to address this type of basic research. These state-of-the-art techniques, together with data from the ongoing genome projects, are likely to produce significant fundamental advances. Projected Timetable and Funding Requirements This requires immediate action. Investigator-initiated grants should be used, but sponsorship by industry for this type of research also should be sought. Develop Tools for Diagnostic and Population Biology Issues (e.g., Screening Programs) Research Goals A combination of programs is recommended, including a basic research program designed to identify new methods for diagnostic and population screening assays and a clinical research program in which these assays can be applied. There are many potential applications of such methods, including: Diagnostic tests for GI diseases in which the diagnosis currently involves invasive procedures or sophisticated equipment and technology, such as celiac disease, glycogen storage diseases, hereditary fructosemia and disorders of fatty acid oxidation Diagnostic tests that can be used in large populations for screening programs and population biology research Diagnostic tests to identify markers of tissue injury, such as fibrosis, cirrhosis, graft rejection and risk for cancer Assays for modifying genes Research Strategies This goal will require basic research to develop diagnostic tools and methods based on DNA, microarray and proteonomic techniques. For example, in cystic fibrosis, genome scanning methods could be used to study the inheritance of microsatellite markers in multigenerational kindreds. The resulting linkage data could lead to identification of additional modifying genes. This goal will also require applied research in the design of screening studies, such as have been undertaken for Tay-Sachs disease and α1-antitrypsin deficiency, and population biology studies. Projected Timetable and Funding Requirements Immediate action is recommended via nationally funded peer-reviewed grant mechanisms. We also recommend partnerships with industry to develop diagnostic assays and sponsor screening studies. A planning committee should be convened to identify key areas and to plan large-scale multicenter collaborative efforts for the development of screening programs and population biology studies. For this effort, multiple NIH institutes as well as foundations should be approached for financial support. HEALTH AND ECONOMIC OUTCOMES A greater understanding of the molecular basis of GI disease and improved methods for diagnosis, prophylaxis and treatment will have a major impact on the clinical outcome for affected children and on the associated costs of health care. This section of the Research Agenda has described the potential use of data from genome projects, as well as use of microarray and proteonomic techniques, to achieve these goals. How the recommended research programs will influence health outcome and cost of care is described in the following sections focusing on specific disorders and organ systems.