| 1. |
- Mangano, M. C., et al.
(författare)
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The aquaculture supply chain in the time of covid-19 pandemic : Vulnerability, resilience, solutions and priorities at the global scale
- 2022
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Ingår i: Environmental Science and Policy. - : Elsevier. - 1462-9011 .- 1873-6416. ; 127, s. 98-110
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Tidskriftsartikel (refereegranskat)abstract
- The COVID-19 global pandemic has had severe, unpredictable and synchronous impacts on all levels of perishable food supply chains (PFSC), across multiple sectors and spatial scales. Aquaculture plays a vital and rapidly expanding role in food security, in some cases overtaking wild caught fisheries in the production of high quality animal protein in this PFSC. We performed a rapid global assessment to evaluate the effects of the COVID19 pandemic and related emerging control measures on the aquaculture supply chain. Socio-economic effects of the pandemic were analysed by surveying the perceptions of stakeholders, who were asked to describe potential supply-side disruption, vulnerabilities and resilience patterns along the production pipeline with four main supply chain components: a) hatchery, b) production/processing, c) distribution/logistics and d) market. We also assessed different farming strategies, comparing land-vs. sea-based systems; extensive vs. intensive methods; and with and without integrated multi-trophic aquaculture, IMTA. In addition to evaluating levels and sources of economic distress, interviewees were asked to identify mitigation solutions adopted at local / internal (i.e., farm site) scales, and to express their preference on national / external scale mitigation measures among a set of a priori options. Survey responses identified the potential causes of disruption, ripple effects, sources of food insecurity, and socio-economic conflicts. They also pointed to various levels of mitigation strategies. The collated evidence represents a first baseline useful to address future disaster-driven responses, to reinforce the resilience of the sector and to facilitate the design reconstruction plans and mitigation measures, such as financial aid strategies.
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| 2. |
- Sarà, G., et al.
(författare)
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The Synergistic Impacts of Anthropogenic Stressors and COVID-19 on Aquaculture : A Current Global Perspective
- 2022
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Ingår i: Reviews in Fisheries Science & Aquaculture. - : Informa UK Limited. - 2330-8249 .- 2330-8257. ; 30:1, s. 123-135
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Tidskriftsartikel (refereegranskat)abstract
- The rapid, global spread of COVID-19, and the measures intended to limit or slow its propagation, are having major impacts on diverse sectors of society. Notably, these impacts are occurring in the context of other anthropogenic-driven threats including global climate change. Both anthropogenic stressors and the COVID-19 pandemic represent significant economic challenges to aquaculture systems across the globe, threatening the supply chain of one of the most important sources of animal protein, with potential disproportionate impacts on vulnerable communities. A web survey was conducted in 47 countries in the midst of the COVID-19 pandemic to assess how aquaculture activities have been affected by the pandemic, and to explore how these impacts compare to those from climate change. A positive correlation between the effects of the two categories of drivers was detected, but analysis suggests that the pandemic and the anthropogenic stressors affect different parts of the supply chain. The immediate measurable reported losses varied with aquaculture typology (land vs. marine, and intensive vs. extensive). A comparably lower impact on farmers reporting the use of integrated multitrophic aquaculture (IMTA) methods suggests that IMTA might enhance resilience to multiple stressors by providing different market options under the COVID-19 pandemic. Results emphasize the importance of assessing detrimental effects of COVID-19 under a multiple stressor lens, focusing on areas that have already locally experienced economic loss due to anthropogenic stressors in the last decade. Holistic policies that simultaneously address other ongoing anthropogenic stressors, rather than focusing solely on the acute impacts of COVID-19, are needed to maximize the long-term resilience of the aquaculture sector.
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| 3. |
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| 4. |
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| 5. |
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| 6. |
- Scott, S. M., et al.
(författare)
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Chronic constipation in adults: Contemporary perspectives and clinical challenges. 1: Epidemiology, diagnosis, clinical associations, pathophysiology and investigation
- 2021
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Ingår i: Neurogastroenterology and Motility. - : Wiley. - 1350-1925 .- 1365-2982. ; 33:6
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND Chronic constipation is a prevalent disorder that affects patients' quality of life and consumes resources in healthcare systems worldwide. In clinical practice, it is still considered a challenge as clinicians frequently are unsure as to which treatments to use and when. Over a decade ago, a Neurogastroenterology & Motility journal supplement devoted to the investigation and management of constipation was published (2009; 21 (Suppl.2)). This included seven articles, disseminating all themes covered during a preceding 2-day meeting held in London, entitled "Current perspectives in chronic constipation: a scientific and clinical symposium." In October 2018, the 3rd London Masterclass, entitled "Contemporary management of constipation" was held, again over 2 days. All faculty members were invited to author two new review articles, which represent a collective synthesis of talks presented and discussions held during this meeting. PURPOSE This article represents the first of these reviews, addressing epidemiology, diagnosis, clinical associations, pathophysiology, and investigation. Clearly, not all aspects of the condition can be covered in adequate detail; hence, there is a focus on particular "hot topics" and themes that are of contemporary interest. The second review addresses management of chronic constipation, covering behavioral, conservative, medical, and surgical therapies.
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| 7. |
- Haas, Brian J., et al.
(författare)
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Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans
- 2009
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 461:7262, s. 393-398
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Tidskriftsartikel (refereegranskat)abstract
- Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement(1). To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population(1). Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion(2). Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars(3,4). Here we report the sequence of the P. infestans genome, which at similar to 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for similar to 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.
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| 8. |
- Wang, Sophia S., et al.
(författare)
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HLA Class I and II Diversity Contributes to the Etiologic Heterogeneity of Non-Hodgkin Lymphoma Subtypes
- 2018
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 78:14, s. 4086-4096
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Tidskriftsartikel (refereegranskat)abstract
- A growing number of loci within the human leukocyte antigen (HLA) region have been implicated in non-Hodgkin lymphoma (NHL) etiology. Here, we test a complementary hypothesis of "heterozygote advantage" regarding the role of HLA and NHL, whereby HLA diversity is beneficial and homozygous HLA loci are associated with increased disease risk. HLA alleles at class I and II loci were imputed from genome-wide association studies (GWAS) using SNP2HLA for 3,617 diffuse large B-cell lymphomas (DLBCL), 2,686 follicular lymphomas (FL), 2,878 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL), 741 marginal zone lymphomas (MZL), and 8,753 controls of European descent. Both DLBCL and MZL risk were elevated with homozygosity at class I HLA-B and -C loci (OR DLBCL = 1.31, 95% CI = 1.06-1.60; OR MZL = 1.45, 95% CI = 1.12-1.89) and class II HLA-DRB1 locus (OR DLBCL = 2.10, 95% CI = 1.24-3.55; OR MZL = 2.10, 95% CI = 0.99-4.45). Increased FL risk was observed with the overall increase in number of homozygous HLA class II loci (P trend < 0.0001, FDR = 0.0005). These results support a role for HLA zygosity in NHL etiology and suggests that distinct immune pathways may underly the etiology of the different NHL subtypes. Significance: HLA gene diversity reduces risk for non-Hodgkin lymphoma.
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| 9. |
- Cameron-McDermott, Suzette M., et al.
(författare)
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Antimicrobial susceptibility of Neisseria gonorrhoeae isolates and syndromic treatment of men with urethral discharge in Kingston, Jamaica, 2018-19
- 2022
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 77:1, s. 218-222
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: To quantitatively determine the antimicrobial susceptibility of clinical Neisseria gonorrhoeae isolates from men with urethral discharge in Jamaica and to describe the syndromic treatment therapies administered.METHODS: Urethral eSwabs (Copan) were collected from 175 men presenting with urethral discharge to the Comprehensive Health Centre STI Clinic, Kingston, Jamaica. Clinical information was collected and MICs of eight antimicrobials were determined for N. gonorrhoeae isolates (n = 96) using Etest and interpreted using CLSI criteria.RESULTS: The median age of the subjects was 28 years (range: 18-73 years) with a median of 2 sexual partners (range: 1-25) per male in the previous 3 months. All examined N. gonorrhoeae isolates were susceptible to ceftriaxone (96/96), azithromycin (91/91), cefixime (91/91) and spectinomycin (91/91). For ciprofloxacin and gentamicin, respectively, 98.9% (91/92) and 91.3% (84/92) of the isolates were susceptible and 1.1% (1/92) and 8.7% (8/92) showed intermediate susceptibility/resistance. For tetracycline and benzylpenicillin, respectively, 38.0% (35/92) and 22.0% (20/91) of the isolates were susceptible, 52.2% (48/92) and 74.7% (68/91) showed intermediate susceptibility/resistance and 9.8% (9/92) and 3.3% (3/91) were resistant. Syndromic treatment was administered as follows: 93.1% received 250 mg of ceftriaxone intramuscularly plus 100 mg of doxycycline orally q12h for 1-2 weeks and 6.9% received 500 mg of ciprofloxacin orally plus 100 mg of doxycycline orally q12h for 1 week.CONCLUSIONS: Ceftriaxone (250 mg) remains appropriate for gonorrhoea treatment in the examined population of men in Kingston, Jamaica. Surveillance of N. gonorrhoeae AMR should be expanded in Jamaica and other Caribbean countries to guide evidence-based treatment guidelines.
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| 10. |
- Adamczyk, Barbara, 1985, et al.
(författare)
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Pregnancy-Associated Changes of IgG and Serum N-Glycosylation in Camel (Camelus dromedarius).
- 2016
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Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 15:9, s. 3255-65
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Tidskriftsartikel (refereegranskat)abstract
- The dromedary camel (Camelus dromedarius) is an agriculturally important species of high economic value but of low reproductive efficiency. Serum and IgG N-glycosylation are affected by physiological and pathogenic changes and might therefore be a useful diagnostic tool in camel livestock management. This study presents the first comprehensive annotation of the N-glycome from dromedary camel serum as well as their single-domain and conventional antibodies and its subsequent application for camel pregnancy diagnostics. N-glycans were released by PNGaseF, labeled with 2-aminobenzamide (2-AB), and analyzed by hydrophilic interaction liquid chromatography with fluorescent detection (HILIC-UPLC-FLD), enzymatic sequencing and mass spectrometry (MS). The use of a high-throughput robotic platform for sample preparation allowed the rapid generation of glycomics data from pregnant (n = 8) and nonpregnant (n = 8) camels of the Majaheem and Wadha breed. IgG N-glycans dominate the glycan profile of camel serum and present a mixture of core-fucosylated and noncore-fucosylated N-glycans which can contain N-glycolylneuraminic and N-acetylneuraminic acid. Significant pregnancy-associated but breed-independent increases in galactosylation, core-fucosylation, sialylation, and decreases in serum O-acetylation were observed. The monitoring of IgG and serum N-glycosylation presents an attractive complementary test for camel pregnancy diagnostics and presents an interesting tool for biomarker discovery in camel health and breeding.
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| 11. |
- Carrington, G., et al.
(författare)
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Human skeletal myopathy myosin mutations disrupt myosin head sequestration
- 2023
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Ingår i: JCI Insight. - 2379-3708. ; 8:21
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Tidskriftsartikel (refereegranskat)abstract
- Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.
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| 12. |
- El-Sayed, Najib M., et al.
(författare)
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The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.
- 2005
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 309:5733, s. 409-15
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Tidskriftsartikel (refereegranskat)abstract
- Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.
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| 13. |
- Schwarz, J., et al.
(författare)
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F8 haplotype and inhibitor risk: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort
- 2013
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Ingår i: Haemophilia. - : Wiley. - 1351-8216. ; 19:1, s. 113-118
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Tidskriftsartikel (refereegranskat)abstract
- Ancestral background, specifically African descent, confers higher risk for development of inhibitory antibodies to factor VIII (FVIII) in haemophilia A. It has been suggested that differences in the distribution of FVIII gene (F8) haplotypes, and mismatch between endogenous F8 haplotypes and those comprising products used for treatment could contribute to risk. Data from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort were used to determine the association between F8 haplotype 3 (H3) vs. haplotypes 1 and 2 (H1 + H2) and inhibitor risk among individuals of genetically determined African descent. Other variables known to affect inhibitor risk including type of F8 mutation and human leucocyte antigen (HLA) were included in the analysis. A second research question regarding risk related to mismatch in endogenous F8 haplotype and recombinant FVIII products used for treatment was addressed. Haplotype 3 was associated with higher inhibitor risk among those genetically identified (N = 49) as of African ancestry, but the association did not remain significant after adjustment for F8 mutation type and the HLA variables. Among subjects of all racial ancestries enrolled in HIGS who reported early use of recombinant products (N = 223), mismatch in endogenous haplotype and the FVIII proteins constituting the products used did not confer greater risk for inhibitor development. Haplotype 3 was not an independent predictor of inhibitor risk. Furthermore, our findings did not support a higher risk of inhibitors in the presence of a haplotype mismatch between the FVIII molecule infused and that of the individual.
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| 14. |
- Struwe, W. B., et al.
(författare)
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Identification of O-glycan Structures from Chicken Intestinal Mucins Provides Insight into Campylobactor jejuni Pathogenicity
- 2015
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476. ; 14:6, s. 1464-1477
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1-2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sda-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection.
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| 15. |
- Alexander, Steven M., et al.
(författare)
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Examining horizontal and vertical social ties to achieve social-ecological fit in an emerging marine reserve network
- 2017
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Ingår i: Aquatic conservation. - : Wiley. - 1052-7613 .- 1099-0755. ; 27:6, s. 1209-1223
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Tidskriftsartikel (refereegranskat)abstract
- Most MPA networks are designed only with ecological processes in mind to increase their conservation utility. However, since MPA networks often involve large geographic areas, they also affect and involve multiple actors, institutions, and policy sectors. A key challenge when establishing an effective MPA network is to align the social system' with the biophysical MPA network (the ecological system'). This challenge is often denoted as social-ecological fit'. Facilitating collaborative social interactions among various actors and stakeholders (social connectivity) is equally as important as accomplishing ecological connectivity. New analytical approaches are required to effectively examine this social' dimension of fit. An emerging marine reserve network in Jamaica and the recent invasion of Indo-Pacific lionfish are used as a case study to: (1) examine the extent to which horizontal and vertical social ties bring local and national actors together to collaborate, coordinate, and share knowledge; and (2) assess the extent to which different attributes and features of such multilevel social networks may enhance or inhibit particular aspects of social-ecological fit. Findings suggest that multilevel linkages have played the greatest role in relation to enhancing fit in the marine reserve network in the context of the recent lionfish invasion. However, the long-term propensity of the multi-actor and multilevel networks to enhance social-ecological fit is uncertain given the prevalence of weak social ties, lack of a culture of information sharing and collaboration, and limited financial resources.
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| 16. |
- Carrington, M, et al.
(författare)
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Clinical applications of heart rhythm monitoring tools in symptomatic patients and for screening in high-risk groups
- 2022
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Ingår i: Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing, arrhythmias, and cardiac cellular electrophysiology of the European Society of Cardiology. - : Oxford University Press (OUP). - 1532-2092. ; 24:11, s. 1721-1729
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Tidskriftsartikel (refereegranskat)abstract
- Recent technological advances have facilitated and diversified the options available for the diagnosis of cardiac arrhythmias. Ranging from simple resting or exercise electrocardiograms to more sophisticated and expensive smartphones and implantable cardiac monitors. These tests and devices may be used for varying periods of time depending on symptom frequency. The choice of the most appropriate heart rhythm test should be guided by clinical evaluation and optimized following accurate characterization of underlying symptoms, ‘red flags’, risk factors, and consideration of cost-effectiveness of the different tests. This review provides evidence-based guidance for assessing suspected arrhythmia in patients who present with symptoms or in the context of screening, such as atrial fibrillation or advanced conduction disturbances following transcatheter aortic valve implantation in high-risk groups. This is intended to help clinicians choose the most appropriate diagnostic tool to facilitate the management of patients with suspected arrhythmias.
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| 17. |
- Carrington, M, et al.
(författare)
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Monitoring and diagnosis of intermittent arrhythmias: evidence-based guidance and role of novel monitoring strategies
- 2022
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Ingår i: European heart journal open. - : Oxford University Press (OUP). - 2752-4191. ; 2:6, s. oeac072-
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Tidskriftsartikel (refereegranskat)abstract
- Technological advances have made diagnosis of heart rhythm disturbances much easier, with a wide variety of options, including single-lead portable devices, smartphones/watches to sophisticated implantable cardiac monitors, allowing accurate data to be collected over different time periods depending on symptoms frequency. This review provides an overview of the novel and existing heart rhythm testing options, including a description of the supporting evidence for their use. A description of each of the tests is provided, along with discussion of their advantages and limitations. This is intended to help clinicians towards choosing the most appropriate test, thus improving diagnostic yield management of patients with suspected arrhythmias.
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| 18. |
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| 19. |
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| 20. |
- Varshney, Gaurav K., et al.
(författare)
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A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish
- 2016
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 11:12, s. 2357-2375
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Tidskriftsartikel (refereegranskat)abstract
- The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F-1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.
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| 21. |
- Varshney, Gaurav K., et al.
(författare)
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CRISPRz : a database of zebrafish validated sgRNAs
- 2016
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:D1, s. D822-D826
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Tidskriftsartikel (refereegranskat)abstract
- CRISPRz (http://research.nhgri.nih.gov/CRISPRz/) is a database of CRISPR/Cas9 target sequences that have been experimentally validated in zebrafish. Programmable RNA-guided CRISPR/Cas9 has recently emerged as a simple and efficient genome editing method in various cell types and organisms, including zebrafish. Because the technique is so easy and efficient in zebrafish, the most valuable asset is no longer a mutated fish (which has distribution challenges), but rather a CRISPR/Cas9 target sequence to the gene confirmed to have high mutagenic efficiency. With a highly active CRISPR target, a mutant fish can be quickly replicated in any genetic background anywhere in the world. However, sgRNA's vary widely in their activity and models for predicting target activity are imperfect. Thus, it is very useful to collect in one place validated CRISPR target sequences with their relative mutagenic activities. A researcher could then select a target of interest in the database with an expected activity. Here, we report the development of CRISPRz, a database of validated zebrafish CRISPR target sites collected from published sources, as well as from our own in-house large-scale mutagenesis project. CRISPRz can be searched using multiple inputs such as ZFIN IDs, accession number, UniGene ID, or gene symbols from zebrafish, human and mouse.
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| 22. |
- Varshney, Gaurav K., et al.
(författare)
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High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
- 2015
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 25:7, s. 1030-1042
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Tidskriftsartikel (refereegranskat)abstract
- The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F-1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
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| 23. |
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