206 CRISPR/Cas9-MEDIATED REPAIR OF THE NHLRC2 LOCUS IN BEEF CATTLE

Abstract

In cattle, a mutation in the NHL-repeat containing 2 genes causes a heritable abnormality referred to as developmental duplications. Calves homozygous for this mutation are affected with a broad range of phenotypes resulting from abnormal neural crest cell migration, most commonly manifested as polymelia, the presence of additional limbs. This mutation has become highly prevalent in Angus beef cattle, as lines of cattle with high genetic merit have been shown to have an increased allele frequency of the mutation. The mutation has been identified as a single nucleotide polymorphism resulting in a valine to alanine substitution in a highly conserved protein-coding region of the gene. CRISPR/Cas9 genome editing technology has been shown to induce changes in the genome by using a guide RNA to target a specific site paired with a Cas-9 protein to create a break in the DNA. These breaks are repaired by either nonhomologous end joining or homology-directed repair. The aim of this preliminary study was to determine the editing efficiency of CRISPR/Cas-9 proteins paired with site-specific guide RNA using cell lines derived from animals homozygous and heterozygous for the NHLRC2 mutation. Bovine fetal fibroblasts of both genotypes were grown in DMEM/F10 media supplemented with 10% fetal bovine serum, 0.01 µg mL⁻¹ of basic fibroblast growth factor, 1 mL L⁻¹ penicillin streptomycin, and 1 mL L⁻¹ of amphotericin B. Cells were plated in a 6-well plate at 80,000 cells/well 48 h before transfection. Two 20-nucleotide guide RNA targeting the genome near the developmental duplications mutation were designed and ligated into pSpCas9(BB)-2A-GFP CRISPR plasmids. Six microliters of Fugene 6 was added to 150 µL of Opti-MEM followed by 2 µg of plasmid DNA and complexed at 37°C for 15 min before being added to each well. At 24 h after transfection, cells were detached with trypsin and sorted by fluorescence-activated cell sorting. When wells were confluent, DNA was extracted using 65 µL of QuickExtract DNA extraction solution. The 500-bp fragment surrounding the mutation was amplified and subjected to a restriction enzyme digest. Fragments with the exact sequence of the mutation were cleaved, whereas normal genotypes or edited genotypes without the mutation sequence remain uncleaved. Fragments were size separated on a 2% agarose gel. Band intensity under ultraviolet illumination was calculated with GelReader and the ratios of cleaved versus uncleaved fragments were compared with the ratio of control (unedited cell lines) for both guide RNA and both heterozygous and homozygous cell lines. Based on this preliminary data, CRISPRs with guide RNA 1 edited the genome at the target site at 13.3 and 12.2% for heterozygous and homozygous cell lines, respectively, and guide RNA 2 affected the target site at 2.5 and 4.1% for heterozygous and homozygous, respectively. These data show that the designed guide RNA paired with CRISPRs are able to elicit changes at the desired locus. In order to understand what repair mechanisms were employed at these loci, the next step is to subclone and sequence PCR products.