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转录激活样效应核酸酶（TALEN）介导的CLYBL靶向能够增强转基因表达，并一步生成双报告基因人类诱导多能干细胞（iPSC）和神经干细胞（NSC）系。

Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.

作者信息

Cerbini Trevor, Funahashi Ray, Luo Yongquan, Liu Chengyu, Park Kyeyoon, Rao Mahendra, Malik Nasir, Zou Jizhong

机构信息

NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, United States of America.

Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, United States of America.

出版信息

PLoS One. 2015 Jan 14;10(1):e0116032. doi: 10.1371/journal.pone.0116032. eCollection 2015.

DOI:10.1371/journal.pone.0116032
PMID:25587899
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4294658/
Abstract

Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

摘要

靶向基因组工程以强劲表达转基因是基于干细胞的研究和治疗的一项重要方法。尽管设计核酸酶已被用于大幅提高基因编辑效率，但迄今为止，转基因的靶向添加和稳定表达在人类干细胞中仅限于单个基因/位点，且大多是PPP1R12C/AAVS1位点。在此，我们构建了靶向安全港样基因CLYBL的转录激活样效应核酸酶（TALENs），以38%-58%的效率介导报告基因整合，并使用AAVS1-TALENs和CLYBL-TALENs在人类诱导多能干细胞（iPSCs）和神经干细胞（NSCs）的双安全港位点同时敲入多个报告基因。CLYBL-TALEN工程细胞系在自我更新和分化过程中维持了强劲的报告基因表达，并表明与PPP1R12C/AAVS1相比，靶向CLYBL导致更强的转基因表达且对局部基因表达的干扰更小。TALEN介导的CLYBL工程为人类干细胞中的多种基因修饰提供了改善的转基因表达和选择。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/87bd34330bd0/pone.0116032.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/babadce7773f/pone.0116032.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/4ec91b9e19ea/pone.0116032.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/6e32913c510a/pone.0116032.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/a5a48165b39d/pone.0116032.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/87bd34330bd0/pone.0116032.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/babadce7773f/pone.0116032.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/4ec91b9e19ea/pone.0116032.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/6e32913c510a/pone.0116032.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/a5a48165b39d/pone.0116032.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1c00/4294658/87bd34330bd0/pone.0116032.g005.jpg

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