PRJNA392273
Opisthorchis viverrini
Project title: Programmed gene editing knocks out the growth proliferative factor, granulin, of the of the carcinogenic liver fluke, Opisthorchis viverriniPublic descriptionOrganism: Opisthorchis viverriniSize of Genome: 750 MbAuthors: Patpicha Arunsan, Wannaporn Ittiprasert, Christina J. Cochran, Victoria H. Mann, Sujittra Chaiyadet, Banchob Sripa, Neil D. Young, Michael J. Smout, Javier Sotollo, Alex Loukas, Paul J Brindley, and Thewarach LahaYear: 2017Status: UnpublishedType: miSeq_granulin-1 gene editing by CRISPR/Cas 9 in the adult developmental stage of Opisthorchis viverriniPlatform and configuration:MiSEq 2 x 250 bp standardDepth of coverage 20-50x (GWAS or high frequency mutations)Data Analysis: INDEL discovery for fragmented and contiguous amplicon sequencingDescription: The genomic DNA was extracted from the mature Opisthorchis viverrini liver flukes recovered from experimentally infected hamsters following CRISPR/Cas 9 gene editing targeting exon 1 of granulin-1 Ov-grn-1gene, GenBank accession FJ436341, 6,278 nt. The flukes were transfected in vitro with a plasmid constructed from the GeneArt CRISPR Nuclease Vector. Briefly, each worm individually was subjected to square wave electroporation in the presence of 3 micrograms of vector plasmid encoding the 20 nt guide RNA (plasmid sequence 5'-GATTCATCTACAAGTGTTGA) driven by human U6 promotor and Streptococcus pyogenes Cas 9 endonuclease gene driven by CMV promoter. Both Ov-grn-1 gene transcript and protein level were lost to below deetction from 3-21 days following transfection. In order to construct the targeted (amplicon) sequence library for Illumina Next Generation Sequencing (NGS), a 172 bp fragment spanning the predicted double stranded break site of the Ov.grn-1 locus was amplified using genomic DNAs from O. viverrini recovered from the worms at 7 and 21 days after transfection. PCR amplification using GoTaq polymerase was primed with oligonucleotides flanking 1589-1608 nt of Ov-grn-1. Compatible adaptors and barcodes (GeneRead 12-plex adaptor, Qiagen) were ligated to the pooled amplicons using the Amplicon Library Preparation kit (Qiagen), which is compatible with NGS by the Illumina process. The libraries were quantified by Qiaseq Library Quant Assay Kit (Qiagen), after which the NGS was undertaken at GeneWiz. The insertion-deletion (INDEL) profile of the sequenced amplicons was assessed with assistance of the Snap Gene Software's multi-alignment tool to compare the amplicons and wild type (original) sequences of Ov-grn-1.NAME: Ittiprasert, W.EMAIL: wannaporni@gwu.eduLAB: Brindley LabADDR: 2300 Ross Hall, Department of Microbiology, Immunology and Tropical Diseases,Research Center for Neglected Diseases of Poverty,School of Medicine and Hygiene, The George Washington University, Washington D.C., 20037 USAGrant: National Cancer Institute (NCI), US National Institutes of Health (NIH) award number CA164719