Abstract
Pulmonary Arterial Hypertension (PAH) is a rare vascular disorder affecting 1 to 2 cases per million individuals. Monoallelic mutations in the BMPR2 gene are the most common genetic risk factor, with about 20% of carriers affected. BMPR2 is a serine/threonine kinase receptor in the transforming growth factor beta (TGF-β) superfamily. Despite well-known associations with PAH, the role of BMPR2 in the progression of the disorder remains poorly understood due in part to the lack of appropriate preclinical genetic models. The homozygous BMPR2 (-/-) knockout genotype is embryonic lethal, which is an obstacle to creating a suitable genetic biomedical model for PAH. To overcome this barrier, we used a single guide RNA (sgRNA) CRISPR/Cas9 approach to target exon 3 of the ovine BMPR2 gene, in combination with a single-stranded oligo-deoxynucleotide (ssODN) synonymous homology-directed repair (HDR) template to create heterozygous BMPR2 (+/-) sheep. Heterozygote lambs could be generated in two ways with this approach – high editing efficiency coupled with ssODN homology-directed repair of one of the edited alleles, OR intermediate editing efficiency with low to no presence of HDR in the genetic sequence. Our main objectives were to:1.) Determine optimal concentrations of ssODN (100, 300, and 600 ng/ul) to promote HDR and 2.) Identify the sgRNA which maximized the probability of obtaining heterozygous lambs. A ssODN was designed to serve as a synonymous repair template that disrupted the PAM site for a pair of sgRNAs targeting BMPR2 exon 3 on opposite strands. We electroporated ovine zygotes six hours post-fertilization using sgRNA (100 ng/µL)/Cas9 (200 ng/µL) ribonucleoprotein (RNP) complexes alongside three ODN concentrations (100, 300, and 600 ng/µL). Zygotes were cultured for seven days, and blastocysts were lysed, PCR-amplified, and Sanger sequenced. Sequences were analyzed using TIDER (Brinkman et al., Nucl. Acids Res., 2018). TIDER is a computer model which quantifies the frequency of targeted small nucleotide changes introduced by CRISPR in combination with HDR using a donor template. A Kruskal-Wallis test compared the efficacy of HDR at 100, 300, and 600 ng/µL of ODN template. A significant difference was found between groups with 600 ng/µL giving the highest rate of HDR (P < 0.0001). Post-hoc pairwise comparisons using a Dunn's test found that sgRNA1 at 600 ng/ul ODN was significantly more efficient for HDR repair as compared with sgRNA2 (P < 0.05). The editing efficiency of the blastocysts collected using the 600 ng/µLODN and sgRNA1 treatment combination was 44.7% high (>66%), 42.6% intermediate (66-33%), and 8.5% low (< 33%) knockout rate, and 4.2% wild-type. The knockin rate was 27.7% high (>20%), 21.3% intermediate (20-5%), and 51% low to no (< 5%) HDR. These data predict that about one-half the transferred embryos subject to these conditions will have the desired heterozygous genotype [BMPR2 (+/-) sheep].