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

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

Louisville, Kentucky

Congreso; 42nd Annual Conference of the IETS; 2016

The rapid Introductionof engineered nucleases technologies, such as ZFNs, TALENs and CRISPR, providesnew opportunities for edition of genes in a targeted and rather simple fashion.Few reports are available regarding CRISPR efficiency in domestic species. Here,the CRISPR-­‐Cas9 system was employed to develop knock-­‐out and knock-­‐in allelesof the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblastsand in  IVF embryos. Five sgRNAs were designedto target a 875 bp region within prnp exon 3, all five were co-­‐delivered withhCas9 and a homologous recombination vector carrying gfp (pHRegfp) . For cells,two transfection conditions were compared: 2 μg hCas9 + 1 μg sgRNAs mix +/-­‐ 2 μg pHREGFP (1X) versus 4 μg hCas9 + 2 μg sgRNAs mix +/-­‐ 4 μg pHREGFP (2X). For IVF zygotes, cytoplasmicinjection was conducted with two RNA concentrations: a) 50 ng/μl hCas9 RNA + 25 ng/μl sgRNAs mix (RNA1X), +/-­‐50 ng/μl pHREFGP and b) 100 ng/μl hCas9 + 50 ng/μl sgRNAs mix (RNA2X), +/-­‐ 100 ng/μl pHREGFP which were compared to plasmidinjections with 100 ng/μlpCMVCas9 + 50 ng/μl pU6sgRNAsmix (DNA2X), +/-­‐ 100 ng/μl pHREGFP. The pHREGFP was always injected as plasmid, at the same conditionsto hCas9. DNA from cells was subjected to PCR, Surveyor assay and sequence analysis.Embryo analysis was conducted on whole-­‐genome-­‐ amplified DNA from blastocysts,followed by PCR assays and sequencing. In cells, 2X transfection resulted in indelsand amplification of PCR products of lower MW than the wild-­‐type, indicative ofthe deletion of a part of the targeted PRNP region. However, it was not possibleto detect an effect for 1X transfection. For the group transfected with pHREGFP,insertion of a partial EGFP sequence was  detected (383 bp). Regarding embryo injection,higher blastocyst rates were obtained in all groups injected with RNA (Table 1).In 48% (21/43) of the sequenced blastocysts specific gene editing was detected (Table1). Modifications varied among single base pair shift (3/43; 7%), high level ofmismatches all over the targeted sequence and vicinity (12/43; 27.9%), full deletionof the 875 bp region (1/43; 2.3%), and partial insertion of 100-­‐498 bp pHREGFPfragments between the HR arms (5/24; 20.8%). Most of these modifications occurredin a mosaic fashion (76%). Results demonstrate that CRISPR/Cas can be efficientlyapplied for site-­‐specific edition.