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Developmental Sensitivity in Schistosoma mansoni to Puromycin To Establish Drug Selection of Transgenic Schistosomes

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      ABSTRACT

      Schistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and loss-of-function approaches can be expected to precipitate the discovery of novel interventions, and drug selection of transgenic schistosomes would facilitate the establishment of stable lines of engineered parasites. Sensitivity of developmental stages of schistosomes to the aminonucleoside antibiotic puromycin was investigated. For the schistosomulum and sporocyst stages, viability was quantified by fluorescence microscopy following dual staining with fluorescein diacetate and propidium iodine. By 6 days in culture, the 50% lethal concentration (LC 50) for schistosomula was 19 μg/ml whereas the sporocysts were 45-fold more resilient. Puromycin potently inhibited the development of in vitro-laid eggs (LC 50, 68 ng/ml) but was less effective against liver eggs (LC 50, 387 μg/ml). Toxicity for adult stages was evaluated using the xCELLigence-based, real-time motility assay (xWORM), which revealed LC 50s after 48 h of 4.9 and 17.3 μg/ml for male and female schistosomes, respectively. Also, schistosomula transduced with pseudotyped retrovirus encoding the puromycin resistance marker were partially rescued when cultured in the presence of the antibiotic. Together, these findings will facilitate selection on puromycin of transgenic schistosomes and the enrichment of cultures of transgenic eggs and sporocysts to facilitate the establishment of schistosome transgenic lines. Streamlining schistosome transgenesis with drug selection will open new avenues to understand parasite biology and hopefully lead to new interventions for this neglected tropical disease.

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        The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
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          The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics. Copyright © 2014, American Association for the Advancement of Science.
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            Author and article information

            Journal
            Antimicrobial Agents and Chemotherapy
            Antimicrob Agents Chemother
            American Society for Microbiology
            0066-4804
            1098-6596
            August 2018
            July 27 2018
            May 14 2018
            : 62
            : 8
            10.1128/AAC.02568-17
            © 2018
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