Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase From Porcine Endothelial Cells by a CRISPR System.
BACKGROUND
We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH).


METHODS
Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells.


RESULTS
Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01.


CONCLUSIONS
Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs.
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. Electronic address: u377126a@ecs.osaka-u.ac.jp.
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Sakai
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Esaki
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Hasuwa
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Ikawa
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Lo
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Matsuura
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Nakahata
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Zenitani
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Asada
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Maeda
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Eguchi
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Okuyama
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
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Miyagawa
D000818:Animals; D015478:Antigens, Heterophile / Q000378:metabolism; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D042783:Endothelial Cells / Q000276:immunology; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D006899:Mixed Function Oxygenases / Q000172:deficiency* / Q000235:genetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D034421:Sus scrofa; D013552:Swine
D015478:Antigens, Heterophile; D012333:RNA, Messenger; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D019158:N-Acetylneuraminic Acid