115 CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT (CRISPR)/Cas9-DIRECTED INACTIVATION OF PORCINE INTERLEUKIN-1B AS A MODEL TO STUDY ENDOMETRIAL RECEPTIVITY AND CONCEPTUS ATTACHMENT

Abstract

Embryonic losses in livestock range from 20 to 40%, with two-thirds of these losses occurring in the peri-implantation period. An understanding of successful pregnancy establishment in pigs is important for translational research and commercial pig production. Failure of conceptus-maternal communication is a major contributor to this loss, yet the molecular control of this process remains unclear. Engineered nucleases such as the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system enable precise modification of the genome compared with traditional gene targeting. In the present study, we hypothesised that the CRISPR/Cas9 system can be used to efficiently produce genetically modified embryo models in the pig that will answer key questions about the complex molecular dialogue between conceptus and maternal uterus. We focused on interleukin-1β (IL1B), a cytokine believed to contribute to rapid trophoblast elongation and pregnancy establishment in pigs. The pig is unique in having 2 forms of the IL1B gene: the systemic form (IL1B) and the conceptus form (IL1BL) that is expressed only at peri-implantation. The coding sequence of the 2 forms of IL1B share 92% similarity. Our objective was to develop 2 types of cloned swine embryos, each with a CRISPR/Cas9-mediated gene inactivation of either IL1B or ILB1L. To target unique regions of IL1B and IL1BL in our fetal fibroblast somatic cell nuclear transfer (SCNT) donor cells, existing Sus scrofa genome data (10.2 annotation) was combined with donor cell Illumina high-throughput sequencing and Sanger sequencing of PCR products to produce consensus IL1B and IL1BL sequences used for targeted disruption. We developed a CRISPR/Cas9 pipeline to identify potential targets 20 nucleotides in length to disrupt the IL1BL and IL1B start codon, while at the same time screening for any potential off-target matches. The pipeline was able to select a group of 11 CRISPR single-chain guide RNAs (sgRNAs; 6 for IL1B and 5 for IL1BL) from an initial library of 413 sgRNAs. Porcine fibroblasts cultured in DMEM, 15% fetal bovine serum, and 10 mg mL^–1 gentamicin were trypsinized and transferred to electroporation medium (75% cytosalts, 25% Opti-MEM), diluted to 1 million cells in 200 μL, and added to 2 mm gap electroporation cuvettes. Cells received 2 μg of pairs of sgRNAs bracketing the start codon and were electroporated with three 1-ms square wave pulses at 250 V. Transfected cells were resuspended in DMEM and plated at low density (~50 cells/plate). The Surveyor Nuclease assay was used to determine a minimum of 5 unique cleavage products in the target DNA in each gene form for heterogeneous cell populations. Clonal populations of targeted cells are currently being characterised for use in SCNT and embryo transfer to identify the effects of IL1B/IL1BL disruption on key molecular pathways during early development (Days 8, 12, and 18 of gestation).