Programmable Nuclease-Based Integration into Novel Extragenic Genomic Safe Harbor Identified from Korean Population-Based CNV Analysis

Eun-Seo Lee; Sanghoon Moon; Kwaku Dad Abu-Bonsrah; Yun Kyoung Kim; Mi Yeong Hwang; Young Jin Kim; Seokjoong Kim; Nathaniel S Hwang; Hyongbum Henry Kim; Bong-Jo Kim

doi:10.1016/j.omto.2019.07.001

Programmable Nuclease-Based Integration into Novel Extragenic Genomic Safe Harbor Identified from Korean Population-Based CNV Analysis

Mol Ther Oncolytics. 2019 Jul 23:14:253-265. doi: 10.1016/j.omto.2019.07.001. eCollection 2019 Sep 27.

Authors

Eun-Seo Lee^{1

2}, Sanghoon Moon³, Kwaku Dad Abu-Bonsrah⁴, Yun Kyoung Kim³, Mi Yeong Hwang³, Young Jin Kim³, Seokjoong Kim⁵, Nathaniel S Hwang^{2

6

7}, Hyongbum Henry Kim^{1

8

9

10}, Bong-Jo Kim³

Affiliations

¹ Department of Pharmacology, Yonsei University College of Medicine, Seoul 03372, Republic of Korea.
² School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Republic of Korea.
³ Division of Genome Research, Center for Genome Science, Korea National Institute of Health, Chungcheongbuk-do 28159, Korea.
⁴ Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, VIC 3052, Australia.
⁵ ToolGen, Seoul 08501, Republic of Korea.
⁶ Interdisciplinary Program in Bioengineering, Seoul National University, Seoul 08826, Republic of Korea.
⁷ BioMax Institute of Seoul National University, Seoul 08826, Republic of Korea.
⁸ Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul 03372, Republic of Korea.
⁹ Center for Nanomedicine, Institute of Basic Science (IBS), Seoul 03772, Republic of Korea.
¹⁰ Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03372, Republic of Korea.

Abstract

Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.

Keywords: TALEN; copy number variation region; genome editing; safe harbor.