40 KNOCKOUT OF GOAT NUCLEOPORIN 155 (NUP155) GENE USING CRISPR/Cas9 SYSTEMS

Abstract

Nucleoporin 155 (NUP155) is a major component of the nuclear pore complex involved in the cellular nucleo-cytoplasmic transport. NUP155 knockout in mice leads to atrial fibrillation and early sudden cardiac death. Due to the small size of the heart, functional electrophysiology is problematic in transgenic mice. Large transgenic animal models have the potential to offer insights into the role of nuclear pore complex in cardiovascular disease. The goal of this study was to generate NUP155 knockout goats by using the CRISPR/Cas9 systems. Two CRISPR target sites were chosen in the first exon of the goat NUP155 gene and CRISPR knockout vectors were constructed. Surveyor assay showed that the 2 CRISPR vectors efficiently induced a double-strand break with cleavage efficiencies of 30 to 50% in transfected goat fibroblast cells. Single cell-derived clones were obtained by limiting dilution of the transfected cells in 48-well plates. In total, 23 single cell clones were isolated and subjected to Surveyor assay followed by sequencing the PCR products spanning the target site. Five of 23 single cell colonies had heterozygous mutations and one carried biallelic modifications of NUP155. The heterozygous cells will be used to produce NUP155± goats by somatic cell nuclear transfer. In conclusion, we demonstrated that CRISPR/Cas9 systems are highly efficient for gene knockout applications in goats.

This work was supported by the Utah Science Technology and Research Initiative, Utah Multidisciplinary Arrhythmia Consortium and Utah Agricultural Experiment Station project #1100.