Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

LIU Hui; LIU Chang; ZHAO Yu-hang; HAN Xue-jie; ZHOU Zheng-wei; WANG Chen; LI Rong-feng; LI Xue-ling

首页> 中文期刊> 《农业科学学报（英文版）》 >Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

AI论文写作 >>

开具论文收录证明 >>

页面导航

摘要
著录项
引文网络
相关主题

摘要

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene knock-ins with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in bovine and dairy goat fetal fibroblasts.To test the knock-in efficiency,a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin(MSTN)gene at exon 2,while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2.Donor plasmids utilizing the ZFNs,TALENs,and CRISPR/Cas9 cutting sites were constructed in the GFP-PGK-NeoR plasmid background,including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1(hFat-1)or enhanced green fluorescent protein(eGFP).Subsequently,the ZFNs,TALENs,or CRISPR/Cas9 and the hFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts.After G418 (Geneticin) selection,single cells were obtained by mouth pipetting,flow cytometry or a cell shove.The gene knock-in events were screened by PCR across the homologous arms.The results showed that in bovine fetal fibrobalsts,the efficiencies of ZFNs-mediated eGFP and hFat-1 gene knock-ins were 13.68 and 0%,respectively.The efficiencies of CRISPR/Cas9-mediated eGFP and hFat-1 gene knock-ins were 77.02 and 79.01%,respectively.The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system.Additionally,the hFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system.The difference of knock-in efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant (P0.01).In the dairy goat fetal fibroblasts,the efficiencies of TALENs-mediated eGFP and hFat-1 gene knock-ins were 32.35 and 26.47%,respectively.The efficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%,respectively.The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant (P0.01).This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs.The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.

著录项

来源
《农业科学学报（英文版）》 |2018年第2期|406-414|共9页
作者
LIU Hui; LIU Chang; ZHAO Yu-hang; HAN Xue-jie; ZHOU Zheng-wei; WANG Chen; LI Rong-feng; LI Xue-ling;
展开▼
作者单位

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks,Inner Mongolia University,Hohhot 010070,P.R.China;

Research Center for Laboratory Animal Science,Inner Mongolia University,Hohhot 010070,P.R.China;

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks,Inner Mongolia University,Hohhot 010070,P.R.China;

Research Center for Laboratory Animal Science,Inner Mongolia University,Hohhot 010070,P.R.China;

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks,Inner Mongolia University,Hohhot 010070,P.R.China;

Research Center for Laboratory Animal Science,Inner Mongolia University,Hohhot 010070,P.R.China;

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks,Inner Mongolia University,Hohhot 010070,P.R.China;

Research Center for Laboratory Animal Science,Inner Mongolia University,Hohhot 010070,P.R.China;

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks,Inner Mongolia University,Hohhot 010070,P.R.China;

Research Center for Laboratory Animal Science,Inner Mongolia University,Hohhot 010070,P.R.China;

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks,Inner Mongolia University,Hohhot 010070,P.R.China;

Research Center for Laboratory Animal Science,Inner Mongolia University,Hohhot 010070,P.R.China;

State Key Laboratories of Reproductive Medicine,Nanjing Medical University,Nanjing 210029,P.R.China;

Jiangsu Key Laboratory of Xenotransplantation,Nanjing Medical University,Nanjing 210029,P.R.China;

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks,Inner Mongolia University,Hohhot 010070,P.R.China;

Research Center for Laboratory Animal Science,Inner Mongolia University,Hohhot 010070,P.R.China;

展开▼
原文格式 PDF
正文语种 eng
中图分类
关键词

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

摘要

著录项

引文网络

相关主题

期刊订阅