Full
Outline
The purpose of this study was to establish an efficient pig cell gene editing technology system and lay a foundation for the application of PE system in pig genetics and breeding. Multiple candidate sites were selected from the coding region of VIM gene， and sgRNA was designed strictly according to the target site design principle of CRISPR/Cas9 system. The candidate sgRNAs were scored by bioinformatics tools， and the two sgRNA sequences with the highest scores were screened out. Based on the above two sgRNAs， the corresponding pegRNAs were further designed. The cells were treated with drugs hydroxyurea and nocodazole to stay in the G1/S or G2/M phase， and the nCas9-RT fusion protein editor vector and pegRNA expression vector were electro-transfected into the cells. After 48 h， the genomic DNA of the mixed cells was extracted， the target sites were amplified by PCR， and the next-generation sequencing was performed. CRISPResso2 was used to analyze the editing results. The results showed that the editing efficiency of PE2 system in PK15 cells was between 0.16% and 14.03%. The editing efficiency in IPEC-J2 cells was between 0.08% and 0.58%. The editing efficiency of PEmax system in PK15 cells was between 0.63% and 23.25%. However， the editing efficiency of PE4 and PE4max systems in PK15 cells was poor， with the highest editing efficiency of 0.7% and 0.36%， respectively. Regulating the cell cycle could improve the editing efficiency of the PE2 system for the editing types of single base substitution and single base deletion of porcine VIM gene. The editing efficiency of the former had increased by 1.4 times， and the editing efficiency of the latter had increased by 2.6 times. According to the results， PE technology can perform a variety of different types of precise editing of pig VIM gene and improve its editing efficiency through cell cycle regulation. It provides a technical reference for the next step to combine somatic cell cloning technology to prepare pig populations with improved production performance.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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