Senior Research Scientist
Immunology Research Centre
St Vincents Hospital
Targeted transgene insertion in pig cells using a high fidelity CRISPR system
Nella Fisicaro1, Evelyn Salvaris1, Mark Nottle2, Peter Cowan1,3.
1Immunology Research Centre, St Vincents Hospital, Melbourne, Australia; 2Obstetrics and Gynaecology, University of Adelaide, Adelaide, Australia; 3Department of Medicine, University of Melbourne, Melbourne, Australia
Background: Pig xenografts provoke a powerful immune response that is difficult to fully control by immunosuppression alone. To address this problem, genetic modification of the donor pig has been used to reduce antigenicity (e.g. by deleting GGTA1, the gene for the xenoantigen αGal) and to add protective human transgenes. Genome editing using the CRISPR/Cas9 guided nuclease system provides an opportunity to streamline this process by enabling precise mutagenesis in cultured pig cells, which can subsequently be used to generate live pigs by cloning. Up to three genes including GGTA1 have been simultaneously knocked out in pigs using native Cas9 (Li eta l, Xenotransplantation 2014), but this nuclease has the potential disadvantage of a high rate of undesirable off-target events related to its use of a single guide RNA (gRNA). The newer variant Fok1-dCas9 uses 2 gRNAs to improve specificity, but its efficacy in pig cells and its capacity to precisely ‘knock in’ transgenes have not been reported.
Aims: (1) To compare the efficiency of mutating GGTA1 in pig cells using either Fok1-dCas9 or Cas9. (2) To knock a human transgene into GGTA1 in pig cells using Fok1-dCas9. Methods: gRNAs were designed to target pig GGTA1 exon 9, which encodes the catalytic domain of α-1,3-galactosyltransferase. Pig fetal fibroblasts were transfected by nucleofection with expression vectors for the nucleases and gRNAs. The efficiency of GGTA1 mutation was determined at the DNA level by Surveyor nuclease assay, which detects small insertions and deletions, and at the protein level by flow cytometry using IB4 lectin to detect changes in αGal expression. In addition, the targeted region was amplified and sequenced.
Results: In the first study, Fok1-dCas9 used with either of 2 pairs of gRNAs knocked out GGTA1 with similar efficiency to Cas9 used with either of 2 single gRNAs. High levels of mutagenesis were identified by the Surveyor assay, and up to 30% of the cells lost αGal expression (i.e. both GGTA1 alleles targeted) within 3 days of transfection. Sequencing of one clone revealed a 25-bp deletion at the expected target site within exon 9 of GGTA1. In the second (knock in) study, expression vectors for Fok1-dCas9 and one pair of gRNAs were co-transfected with a 12kb construct comprising a human transgene flanked by exon 9 homology arms (0.7kb and 1.0kb). PCR analysis of DNA isolated from pooled stable transfectants indicated the presence of cells with correct insertion of the targeting construct into GGTA1. Work is in progress to isolate clonal αGal-negative cells expressing the integrated transgene. Conclusion: The high fidelity Fok1-dCas9 system can be used to efficiently target GGTA1 in pig cells, and preliminary data suggest that it will also be useful for transgene knock-in. This system thus provides a rapid method to generate precisely modified pigs with desirable characteristics for xenotransplantation.
15:30 - 17:00
|Genetic Engineering; Methodology||Targeted transgene insertion in pig cells using a high fidelity CRISPR system||Room 109|