Project title: Schistosoma mansoni gene editing by CRISPR/Cas 9 targeting omega-1 , exon 1
Public description
Organism: Schistosoma mansoni
Size of Genome: 750 Mb
Authors: Ittiprasert et al. More...
Project title: Schistosoma mansoni gene editing by CRISPR/Cas 9 targeting omega-1 , exon 1
Public description
Organism: Schistosoma mansoni
Size of Genome: 750 Mb
Authors: Ittiprasert et al.
Year: 2017
Status: Unpublished
Type: Sanger Direct Sequencing_omega-1 gene editing by CRISPR/Cas 9 in S. mansoni eggs
Platform and configuration:
Sanger Direct Sequencing
miSeq 2 x 250 bp standard
Depth of coverage 20-50x (GWAS or high frequency mutations)
Data Analysis: Knocked-in (KI) with DNA donor(s) Discovery and/or INDELs into schistosome gene target(s) by direct sequencing and high throughput sequencing
Description:
The genomic DNA was extracted from the multi-organism S. mansoni parasite after CRISPR/Cas 9 gene editing with and without DNA donor(s). Briefly, schistosome parasites were transfected with CRISPR vector containing Cas9 gene and guide RNA targeting gene of interest to study the host-parasite immune response mechanism. Then, after 20-24 hours, the DNA donor(s) either single strand donor or double strand donor were introduced into the parasite by electroporation. At least 48 hours after donor introduction, genomic DNAs were investigated for INDELs or KI fragment, also the gene transcript of interested gene was evaluated. In the case of fluorescent protein KI, we also investigate for inserted fluorescent gene transcript driven by parasite gene promoter by RT-PCR from schistosome RNA as well.
Prior to Sanger direct sequencing or to construct the targeted sequence library for Illuminar Next Generation Sequencing (NGS), we amplified the gene fragment from gene modified parasite using a pair of primer flanking the editing sites. The pooled amplicons were proceeded for direct sequencing using the downstream primer(s) or direct ligated with compatible adaptors and barcodes; GeneRead 12-plex adaptor (Qiagen) that compatible to Illunina NGS by Amplicon Library Preparation kit (Qiagen) for miSeq analysis. The miSeq libraries were quantified by Qiaseq Library Quant Assay Kit (Qiagen), then NGS with GeneWiz. The KI and INDELs were analyzed using SnapGene Software with multi-alignment tool comparing with reference sequences.
NAME: Ittiprasert W. EMAIL: wannaporni@gwu.edu LAB: Brindley Lab. ADDR: 2300 Ross Hall, Department of Microbiology, Immunology and Tropical Diseases
School of Medicine and Hygiene, The George Washington University, Washington D.C. Less...