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      Genetic modification of the diarrheal pathogen Cryptosporidium parvum

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          Abstract

          Recent studies into the global causes of severe diarrhea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrheal pathogen after rotavirus 13 . Diarrheal disease is estimated to be responsible for 10.5% of overall child mortality 4 . Cryptosporidium is also an opportunistic pathogen in the context of HIV-AIDS and organ transplantation 5, 6 . There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger, malnourished children and immunocompromised patients 7, 8 . Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes lack of continuous culture, facile animal models, and molecular genetic tools 3, 9 . Here we describe an experimental framework to genetically modify this important human pathogen. We establish and optimize transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we develop a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium CRISPR/Cas9 system, and in vivo selection for aminoglycoside resistance. We derive reporter parasites suitable for in vitro and in vivo drug screening, and we evaluate the basis of drug susceptibility by gene knock out. We anticipate the ability to genetically engineer the parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection and the role of parasite genes in these processes is now open to rigorous investigation.

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          Most cited references49

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Multiplex genome engineering using CRISPR/Cas systems.

            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study.

              Diarrhoeal diseases cause illness and death among children younger than 5 years in low-income countries. We designed the Global Enteric Multicenter Study (GEMS) to identify the aetiology and population-based burden of paediatric diarrhoeal disease in sub-Saharan Africa and south Asia. The GEMS is a 3-year, prospective, age-stratified, matched case-control study of moderate-to-severe diarrhoea in children aged 0-59 months residing in censused populations at four sites in Africa and three in Asia. We recruited children with moderate-to-severe diarrhoea seeking care at health centres along with one to three randomly selected matched community control children without diarrhoea. From patients with moderate-to-severe diarrhoea and controls, we obtained clinical and epidemiological data, anthropometric measurements, and a faecal sample to identify enteropathogens at enrolment; one follow-up home visit was made about 60 days later to ascertain vital status, clinical outcome, and interval growth. We enrolled 9439 children with moderate-to-severe diarrhoea and 13,129 control children without diarrhoea. By analysing adjusted population attributable fractions, most attributable cases of moderate-to-severe diarrhoea were due to four pathogens: rotavirus, Cryptosporidium, enterotoxigenic Escherichia coli producing heat-stable toxin (ST-ETEC; with or without co-expression of heat-labile enterotoxin), and Shigella. Other pathogens were important in selected sites (eg, Aeromonas, Vibrio cholerae O1, Campylobacter jejuni). Odds of dying during follow-up were 8·5-fold higher in patients with moderate-to-severe diarrhoea than in controls (odd ratio 8·5, 95% CI 5·8-12·5, p<0·0001); most deaths (167 [87·9%]) occurred during the first 2 years of life. Pathogens associated with increased risk of case death were ST-ETEC (hazard ratio [HR] 1·9; 0·99-3·5) and typical enteropathogenic E coli (HR 2·6; 1·6-4·1) in infants aged 0-11 months, and Cryptosporidium (HR 2·3; 1·3-4·3) in toddlers aged 12-23 months. Interventions targeting five pathogens (rotavirus, Shigella, ST-ETEC, Cryptosporidium, typical enteropathogenic E coli) can substantially reduce the burden of moderate-to-severe diarrhoea. New methods and accelerated implementation of existing interventions (rotavirus vaccine and zinc) are needed to prevent disease and improve outcomes. The Bill & Melinda Gates Foundation. Copyright © 2013 Elsevier Ltd. All rights reserved.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                24 June 2015
                15 July 2015
                23 July 2015
                23 January 2016
                : 523
                : 7561
                : 477-480
                Affiliations
                [1 ]Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, GA 30602, U.S.A
                [2 ]Department of Cellular Biology, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, GA 30602, U.S.A
                Author notes
                []To whom correspondence should be addressed: Tel.: 1-706-583-0588; Fax: 1-706-542-3582; striepen@ 123456uga.edu
                [*]

                These authors contributed equally to this study

                Article
                NIHMS700702
                10.1038/nature14651
                4640681
                26176919
                9dda16aa-b531-4274-99b2-7bbdde8816cd

                Reprints and permissions information is available at www.nature.com/reprints.

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