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Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells
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  • Wanliu Peng,
  • MengYu Gao,
  • Xinglong Zhu,
  • Xinmei Liu,
  • Guang Yang,
  • Shun Li,
  • Yong Liu,
  • Lang Bai,
  • Jiayin Yang,
  • Ji Bao
Wanliu Peng
West China Hospital of Sichuan University
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MengYu Gao
West China Hospital of Sichuan University
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Xinglong Zhu
West China Hospital of Sichuan University
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Xinmei Liu
West China Hospital of Sichuan University
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Guang Yang
West China Hospital of Sichuan University
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Shun Li
University of Electronic Science and Technology of China
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Yong Liu
West China Hospital of Sichuan University
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Lang Bai
West China Hospital of Sichuan University
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Jiayin Yang
West China Hospital of Sichuan University
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Ji Bao
West China Hospital of Sichuan University

Corresponding Author:[email protected]

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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.
14 Mar 2024Assigned to Editor
14 Mar 2024Submission Checks Completed
14 Mar 2024Reviewer(s) Assigned
19 Mar 2024Review(s) Completed, Editorial Evaluation Pending