School of Medicine and Health Sciences Poster Presentations
Genome Editing for Functional Genomics of Schistosomes
Poster Number
24
Document Type
Poster
Publication Date
3-2016
Abstract
The Streptococcus pyogenes Type II CRISPR system is the keystone of the CRISPR revolution. The system centers on a programmable endonuclease that catalyzes a double stranded break (DSB) in target DNA. The system has been shown to be active in many species including human, mouse, zebra fish, fruit fly, malarial parasite and yeast. It has revolutionized experimental genome editing, and portends hitherto unparalleled advances and positive prospects for gene therapy, biomedicine, and biological systems at large. Adaption of CRISPR technology for editing the genome of schistosomes and other parasitic platyhelminths would be desirable. Here we targeted the IPSE gene of Schistosoma mansoni for ‘knockout’ - deletion mutation in the coding region of the gene. First, using a double reporter plasmid system, NIH 3T3 fibroblasts were transfected with pX330-IPSE1 and pRGS-tgt-IPSE1. By FACS, ~9% cells were RFP+ve, GFP+ve, indicating cleavage of exon 1 of SmIPSE gene (within pRGS-tgt-IPSE1). Second, in vitro incubation of plasmid pRGS-tgt-SmIPSE1 that includes part of exon 1 of the IPSE gene, including a protospacer adjacent motif (PAM), with a macromolecular complex of guide RNA (gRNA) and recombinant Cas9 linearized the plasmid, presumably the consequence of a directed DSB catalyzed by Cas9. Third, cultured schistosomula were transfected using square wave electroporation with recombinant Cas9 of S. pyogenes complexed with gRNA matching residues 22 - 44 of exon 1 of the IPSE gene. Indels at the IPSE locus were evident by two hours later, detected by quantitative PCR, in ~13% of the cells of the parasites. The Type II Cas9 System is active in schistosomes.
Genome Editing for Functional Genomics of Schistosomes
The Streptococcus pyogenes Type II CRISPR system is the keystone of the CRISPR revolution. The system centers on a programmable endonuclease that catalyzes a double stranded break (DSB) in target DNA. The system has been shown to be active in many species including human, mouse, zebra fish, fruit fly, malarial parasite and yeast. It has revolutionized experimental genome editing, and portends hitherto unparalleled advances and positive prospects for gene therapy, biomedicine, and biological systems at large. Adaption of CRISPR technology for editing the genome of schistosomes and other parasitic platyhelminths would be desirable. Here we targeted the IPSE gene of Schistosoma mansoni for ‘knockout’ - deletion mutation in the coding region of the gene. First, using a double reporter plasmid system, NIH 3T3 fibroblasts were transfected with pX330-IPSE1 and pRGS-tgt-IPSE1. By FACS, ~9% cells were RFP+ve, GFP+ve, indicating cleavage of exon 1 of SmIPSE gene (within pRGS-tgt-IPSE1). Second, in vitro incubation of plasmid pRGS-tgt-SmIPSE1 that includes part of exon 1 of the IPSE gene, including a protospacer adjacent motif (PAM), with a macromolecular complex of guide RNA (gRNA) and recombinant Cas9 linearized the plasmid, presumably the consequence of a directed DSB catalyzed by Cas9. Third, cultured schistosomula were transfected using square wave electroporation with recombinant Cas9 of S. pyogenes complexed with gRNA matching residues 22 - 44 of exon 1 of the IPSE gene. Indels at the IPSE locus were evident by two hours later, detected by quantitative PCR, in ~13% of the cells of the parasites. The Type II Cas9 System is active in schistosomes.
Comments
Presented at: GW Research Days 2016.