80 Biallelic CRISPR-Cas9 editing of gene associated with coat colour in microinjected bovine zygotes reaching the blastocyst stage

Abstract

Successful genome editing of blastocysts using zygote microinjection with transcription activator-like effector nucleases has already been accomplished in cattle as well as a limited number of CRISPR-Cas9 microinjections of zygotes, mostly using RNA. Recent editing of the Pou5f1 gene in bovine blastocysts using CRISPR-Cas9, clarifying its role in embryo development, supports the viability of this technology to produce genome edited cattle founders. To further this aim, we hypothesise that editing of the coatomer subunit α (COPA) gene, a protein carrier associated with the dominant red coat colour phenotype in Holstein cattle, is feasible through zygote microinjection. Here, we report successful gene editing of COPA in cattle zygotes reaching the blastocyst stage, a necessary step in creating genome edited founder animals. A single guide RNA was designed to target the sixth exon of COPA. Presumptive zygotes derived from slaughterhouse oocytes by in vitro maturation and fertilization were microinjected either with the PX458 plasmid (Addgene #48138; n=585, 25ng µL−1) or with a ribonucleoprotein effector complex (n=705, 20, 50, 100, and 200ng µL−1) targeting the sixth exon of COPA. Plasmid injected zygotes were selected for green fluorescent protein (GFP) fluorescence at Day 8, whereas protein injected zygotes were selected within 24h post-injection based on ATTO-550 fluorescence. To assess gene editing rates, single Day 8 blastocysts were PCR amplified and screened using the T7 endonuclease assay. Positive structures were Sanger sequenced using bacterial cloning. For plasmid injected groups, the Day 8 blastocyst rate averaged 30.3% (control 18.1%). The fluorescence rate at Day 8 was 6.3%, with a GFP positive blastocyst rate of 1.6%, totaling 7 blastocysts. The T7 assay revealed editing in GFP negative blastocysts and morulae as well, indicating that GFP is not a precise selection tool for successful editing. In protein injection groups, the highest concentration yielded the lowest survival rates (200ng µL−1, 50.0%, n=126), whereas the lowest concentration had the highest survival rate (20ng µL−1, 79.5%, n=314). The Day 8 blastocyst rate reached an average of 25% across groups. However, no edited blastocysts were observed in the higher concentration groups (100,200ng µL−1). The highest number of edited embryos was found in the lowest concentration injected (20ng µL−1, 4/56). Edited embryos showed multiple editing events neighbouring the guide RNA target site ranging from a 12-bp insertion to a 9-bp deletion, as well as unedited sequences. Additionally, one embryo showed a biallelic 15-bp deletion of COPA (10 clones). One possible reason for the presence of only mosaic editing and this in-frame deletion could be that a working copy of COPA is needed for proper blastocyst formation and that a knockout could be lethal. Additional validation and optimization is needed to elucidate the functional role of COPA during early development and its modulation when creating founder animals.