清脆的
基因组编辑
计算机科学
Cas9
计算生物学
图像编辑
生物
遗传学
人工智能
基因
图像(数学)
作者
Wanliu Peng,Mengyu Gao,Xia Zhu,Xinmei Liu,Guang Yang,Shun Li,Yong Liu,Lang Bai,Jiayin Yang,Ji Bao
标识
DOI:10.1002/biot.202300691
摘要
Abstract CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene‐edited pigs. The inefficiency in acquiring gene‐edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene‐edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.
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