Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system

BEVACQUA, R.J.; FERNÁNDEZ-MARTÍN, R.; SAVY, V.; CANEL, N.G.; GISMONDI, M.I.; KUES, W.; CARLSON, D.F.; FAHRENKRUG, S.C.; NIEMANN, H.; FERRARIS, S.; SALAMONE, D.F.

Louisville, Kentucky

Congreso; 42 Annual Conference of the International Embryo Transfer Society; 2016

Until a few years ago, site-specific manipulation of mammal cells and embryos constituted a formidable challenge. The introduction of engineered nucleases technologies, such as ZFNs, TALENs and CRISPR, opened the possibilities to the edition of genes in a specific and simple fashion. However, few reports are available regarding their efficiency on domestic species. In this work, the CRISPR-Cas9 system was tested to knock-out and knock-in bovine prnp gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. To this aim, 5 sgRNAs targeting both ends of a 875 bp region over prnp exon 3, hCas9 and an homologous recombination vector carrying gfp flanked by homology arms contiguous to the sgRNAs target sites (pHRegfp) were used. For cells, two transfection conditions were compared: 2 ug Cas9 + 1 ug sgRNAs mix +/- 2 ug pHRgfp (1X) versus 4 ug Cas9 + 2 ug sgRNAs mix +/- 4 ug pHRgfp (2X). For IVF zygotes, cytoplasmic injection with two RNA concentrations: 50 ng/ul hCas9 RNA + 25 ng/ul sgRNAs mix, +/- 50 ng/ul pHRgfp (1XCrCasRNA) and 100 ng/ul Cas9 + 50 ng/ul sgRNAs mix +/- 100 ng/ul pHRgfp (2X CrCasRNA) were compared to plasmids injection at 100 ng/ul pCMVCas9 + 50 ng/ul pU6sgRNAs mix +/- 100 ng/ul pHRgfp (2X CrCasDNA). The pHRegfp was always injected as plasmid. Cells were subjected to PCR, Surveyor assay and TOPO clones sequencing. Blastocysts´ DNA was amplified and subjected to PCR and sequencing. In cells, 2X transfection resulted in indels and amplification of PCR products of lower MW than the wild type, indicative of deletion of part of the targeted prnp region. However, it was not possible to detect an effect for 1X transfection. For the group injected with pHRgfp, it was possible to detect integration of a 383 bp gfp fragment. Regarding embryo injection, the higher blastocysts rates were obtained for the groups injected with RNA [29/68 (42.6); 20/37 (54.1); 6/19 (31.6); 20/55 (36.4); 26/140 (18.6) and 18/110 (16.4) for 1XCrCasRNA, 1XCrCasRNA+pHRgfp, 2XCrCasRNA, 2X CrCasRNA +pHRgfp, 2XCrCasDNA, 2XCrCasDNA+pHRgfp respectively, p>0.05]. For the groups also injected with pHRegfp, egfp blastocysts rates were higher for 2X CrCasRNA and 2XCrCasDNA than for 1XCrCasRNA [20/20 (100); 17/18 (94); 11/20 (55) respectively]. Specific gene edition of the prnp targeted area was detected in 48% (21/43) of sequenced blastocysts. Modifications varied among single base pair shift (3/43; 7%), high level of mismatches all over the targeted sequence and vicinity (12/43; 27.9%), full deletion of the 875 bp region (1/43; 2.3%), and insertion of 100-498 bp pHRegfp fragments between the HR arms (5/24; 20.8%). All the conditions tested resulted in gene edition [3/9 (33%), 6/10 (60%), 3/6 (50%), 3/8 (37%), 1/6 (16%), 5/10 (50%) for 1XCrCasRNA, 1XCrCasRNA+pHRgfp, 2XCrCasRNA, 2X CrCasRNA+pHRgfp, 2XCrCasDNA, 2XCrCasDNA+pHRgfp respectively]. Our results demonstrate that this new technology can be efficiently applied to site-specific modification of domestic species genomes.