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