NDLI: Dual-reporter surrogate systems for efficient enrichment of genetically modified cells

Content Provider	Springer Nature Link
Author	Ren, Chonghua Xu, Kun Liu, Zhongtian Shen, Juncen Han, Furong Chen, Zhilong Zhang, Zhiying
Copyright Year	2015
Abstract	Isolation of genetically modified cells generated by designed nucleases are challenging, since they are often phenotypically indistinguishable from their parental cells. To efficiently enrich genetically modified cells, we developed two dual-reporter surrogate systems, namely NHEJ-RPG and SSA-RPG based on NHEJ and SSA repair mechanisms, respectively. Repair and enrichment efficiencies of these two systems were compared using different nucleases. In both CRISPR-Cas9- and ZFNs-induced DSB repair studies, we found that the efficiency and sensitivity of the SSA-RPG reporter with direct repeat length more than 200 bp were much higher than the NHEJ-RPG reporter. By utilizing the SSA-RPG reporter, we achieved the enrichment for indels in several endogenous loci with 6.3- to 34.8-fold of non-selected cells. Thus, the highly sensitive SSA-RPG reporter can be used for activity validation of designed nucleases and efficient enrichment of genetically modified cells. Besides, our systems offer alternative enrichment choices either by puromycin selection or FACS.
Starting Page	2763
Ending Page	2772
Page Count	10
File Format	PDF
ISSN	1420682X
Journal	Cellular and Molecular Life Sciences
Volume Number	72
Issue Number	14
e-ISSN	14209071
Language	English
Publisher	Springer Basel
Publisher Date	2015-03-01
Publisher Place	Basel
Access Restriction	Subscribed
Subject Keyword	Surrogate reporter Single-strand annealing Non-homologous end joining DNA repair Genome editing Cell Biology Biomedicine general Life Sciences Biochemistry
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Molecular Medicine Pharmacology Cellular and Molecular Neuroscience

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