利用CRISPR系统敲除猪内皮细胞中的胞苷单磷酸-N-乙酰神经氨酸（CMP-NeuAc）羟化酶

Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase From Porcine Endothelial Cells by a CRISPR System.

作者信息

Sakai R, Esaki Y, Hasuwa H, Ikawa M, Lo P, Matsuura R, Nakahata K, Zenitani M, Asada M, Maeda A, Eguchi H, Okuyama H, Miyagawa S

机构信息

Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

出版信息

Transplant Proc. 2016 May;48(4):1320-2. doi: 10.1016/j.transproceed.2015.10.065.

DOI:10.1016/j.transproceed.2015.10.065

PMID:27320613

Abstract

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.

摘要

背景

我们试图通过使用针对猪胞苷单磷酸-N-乙酰神经氨酸羟化酶（CMAH）的CRISPR（成簇规律间隔短回文重复序列）/Cas9系统来敲除汉努齐乌-戴歇尔（H-D）抗原的表达。

方法

通过将寡核苷酸连接到pX330的BbsI位点，制备用于pCMAH的表达hCas9和sgRNA的质粒。对重叠482 bp的N端和C端EGFP编码区进行PCR扩增，并置于普遍存在的CAG启动子之下。将包含pCMAH的sgRNA靶序列的约400 bp基因组片段置于由EGFP片段侧翼的多克隆位点中。将pCAG-EGxxFP-靶标与带有/不带有sgRNA序列的pX330混合，然后导入HEK293T细胞。