Gene editing in breeding

Gene editing of Porcine Intestinal Organoids: A Next-Generation in vitro Model for Phenotyping

---

Abstract

Porcine intestinal organoids provide an in vitro model to study feed efficiency, nutrient absorption and host-pathogen interactions (Kar et al., 2021). For instance, nutrient absorption of organoids derived from pigs with low and high feed efficiency breeding values can be compared to further investigate the genetic components of these traits. Once potential key genes and/or their genetic variation have been identified, tools such as gene editing can help to understand and validate the role of these genes (Driehuis. & Clevers., 2017). The aim of the study was to develop a standard operating protocol (SOP) for gene editing porcine intestinal organoids with CRISPR-Cas9 and further target a gene that may be relevant for nutrient absorption. First, two different electroporators were tested; the NEPA-porator and the BioRad Gene pulser. The dissociated organoids were transfected with 4.4 kDa TRITC-Dextran in either Opti-MEM or RPMI media. The settings 25ms at 100V (NEPA-porator) versus 15ms at 160V (Gene pulser) were compared. Cell viability of 72% and transfection efficiencies of 20% for 15ms at 160V in Opti-MEM media were found to be most sufficient. Based on these settings the organoids were transfected to target the safe-harbor locus porcine Rosa26 (Sus Scrofa 11.1 NC_010455.5). Three different guide RNAs (gRNAs) were tested for their gene editing efficiencies, gRNA2 was previously published by Xie et.al., (2017). The gene editors were transfected as Ribonucleoprotein complexes (4µM) with a high-specificity Cas9 variant. Transfection with gRNA3 was shown to be most successful with a knock-out (KO) score of 77% based on ICE-analysis (Synthego), whereas the KO score for gRNA2 was 31% and no mutations for gRNA1. The results showed that with the established SOP it is possible to successfully edit porcine intestinal organoids. Next, two gRNAs were designed to target Zinc transporter ZIP4 which is encoded by SLC39A4 and is required for zinc absorption in the intestines. Though, zinc is an essential trace mineral and used as feed additive to support metabolic processes, zinc emissions from livestock farming have a major environmental impact on soil and contribute to antimicrobial resistance. The EU implemented restrictions on the usage of zinc oxide in livestock production. Therefore, identifying genes and genetic variations in relation to zinc absorption, can support pig breeding strategies to improve zinc utilization. The gRNAs were designed to introduce a 704bp deletion between exon 5 and exon 6. PCR amplification of the cell population revealed genotypes for both wild-type (WT) (962 bp) and gene edited alleles (258 bp) on gel electrophoresis. Further DNA analysis will reveal how effective the gRNAs were to induce deletions. Currently, single cell clones are being established to generate pure cell lines with the desired KO. Once established, zinc transport in WT and KO lines will be studied.

Keywords: 2026

How to Cite:

Söllner, J., de Wit, A., van der Valk, E., Kruijt, L., Verstringe, S., Kar, S. & Ellen, E., (2026) “Gene editing of Porcine Intestinal Organoids: A Next-Generation in vitro Model for Phenotyping”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2284542. doi: https://doi.org/10.31274/wcgalp.23583

Rights: 1