Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells

CRISPR/Cas9 technology in conjunction with somatic cell nuclear transplantation (SCNT) provides the primary approach to producing gene-edited pigs, and targeting nuclear donors with CRISPR/Cas9 is crucial. Gene-edited nuclear donors are inefficient due to poor editing efficiency and low delivery efficiency, which are highly associated with CRISPR/Cas9 form selection. Nevertheless, there is not a straightforward method to evaluate CRISPR/Cas9 editing efficiency on the porcine genome. In this study, a fluorescence report signal and micropattern arrays-based platform was developed to visually assess the efficiency of CRISPR/Cas9 editing. Based on the quantity and state of cells grown on micropattern arrays, 200 μm in diameter and 150 μm in spacing were optimal specifications for culturing porcine cells. The editing efficiency of three different CRISPR/Cas9 system forms: DNA, mRNA, and Ribonucleoprotein (RNP) were rapidly evaluated using this platform, with mRNA proving the most effective. Subsequently, four homozygotes with β4GalNT2 gene knockout were quickly obtained by mRNA-based form, which lays the groundwork for the subsequent generation of gene-edited pigs. This platform makes gene knockout efficiency evaluation rapid, intuitive, and efficient. It also holds great promise for customizing evaluation platforms for different cell types, evaluating delivery techniques, and swiftly testing innovative gene editing tools.