243 EFFICIENT EDITION OF THE BOVINE PRNP PRION GENE IN SOMATIC CELLS AND IVF EMBRYOS USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 SYSTEM

Abstract

The rapid introduction of engineered nucleases technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR), provides new opportunities for editing 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 knockout and knock-in alleles of the bovine PRNP gene, responsible for bovine spongiform encephalopathy (mad cow disease), both in bovine fetal fibroblasts and in IVF embryos. Five sgRNAs were designed to target a 875-bp region within prnp exon 3; all 5 were co-delivered with hCas9 and a homologous recombination vector carrying gfp (pHRegfp). For cells, 3 transfection conditions were compared: 2 μg of hCas9 + 1 μg of sgRNAs mix ± 2 μg pHREGFP (1X) versus 4 μg of hCas9 + 2 μg of sgRNAs mix ± 4 μg of pHREGFP (2X). For IVF zygotes, cytoplasmic injection was conducted with 2 RNA concentrations: (a) 50 ng μL–1 hCas9 RNA + 25 ng μL–1 sgRNAs mix (RNA1X), ±50 ng μL–1 pHREGFP, and (b) 100 ng μL–1 hCas9 + 50 ng μL–1 sgRNAs mix (RNA2X), ±100 ng μL–1 pHREGFP, which were compared with plasmid injections with 100 ng μL–1 pCMVCas9 + 50 ng μL–1 pU6sgRNAs mix (DNA2X), ±100 ng μL–1 pHREGFP. The pHREGFP was always injected as plasmid, under the same conditions as 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 indels and amplification of PCR products of lower MW than the wild-type, indicative of the deletion of a part of the targeted PRNP region. However, it was not possible to 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 (Table 1). 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 partial insertion of 100–498 bp pHREGFP fragments between the HR arms (5/24; 20.8%). Most of these modifications occurred in a mosaic fashion (76%). Results demonstrate that CRISPR/Cas can be efficiently applied for site-specific edition of domestic species genomes. Table 1.In vitro development and gene editing efficiency of embryos injected with plasmids or RNA coding for CRISPR/Cas9 system targeting PRNP