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Title of Journal: Biotechnol Lett

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Abbravation: Biotechnology Letters

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Springer Netherlands

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DOI

10.1016/0002-9149(80)90513-5

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1573-6776

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A simple and rapid approach to manipulate pseudora

Authors: Aotian Xu Chao Qin Yue Lang Mingyue Wang Mengyang Lin Chuang Li Rui Zhang Jun Tang
Publish Date: 2015/02/28
Volume: 37, Issue: 6, Pages: 1265-1272
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Abstract

We show that up to 100  viral gene disrupting efficiency was achieved by simple cotransfection of the purified PRV genomes with the clustered regularlyinterspaced short palindromic repeats CRISPR/CRISPRassociated protein 9 Cas9 into cells Furthermore CRISPR/Cas9mediated knockin of 4kblong DNA cassettes into the PRV genome at a positive rate of 50  by a homologyindependent DNA repair mechanism without constructing homology arms This approach requires only a simple plasmid construction and is applicable to knockin of other foreign genesWe thank Dr Zhengfan Jiang at Peking University and Dr Zhongde Wang at Utah State University for generously providing reagents and critically reading the manuscript respectively This work was supported by the research fund from the State Key Laboratory of Agrobiotechnology of China 2015SKLAB612 and the Scientific Research Foundation for the Returned Overseas Chinese Scholars State Education MinistrySupplementary Fig 1—Analysis of the specificity of the antibody produced US3 EP0 and UL50 A Western blot analysis of FLAGUS3 expressed in 293T cells Cell lysates 293T cells transfected with plasmid expressing FLAGUS3 or FLAGvector were probed with the mouse antibody left panel and then reprobed with rabbit antiFLAG antibody right panel B Western blot analysis of FLAGEP0 expressed in 293T cells Cell lysates 293T cells transfected with plasmid expressing FLAGEP0 or FLAGvector were probed with the mouse antibody left panel and then reprobed with rabbit antiFLAG antibody right panel C Western blot analysis of FLAGUL50 expressed in293T cells Cell lysates 293T cells transfected with plasmid expressing FLAGUL50 or FLAGvector were probed with the mouse antibody left panel and then reprobed with rabbit antiFLAG antibody right panel

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