caa a22 4500 606176438 CHVBK 20210128100747.0 cr unu---uuuuu 210128e20150701xx s 000 0 eng 10.1007/s00018-015-1874-6 doi (NATIONALLICENCE)springer-10.1007/s00018-015-1874-6 Dual-reporter surrogate systems for efficient enrichment of genetically modified cells [Elektronische Daten] [Chonghua Ren, Kun Xu, Zhongtian Liu, Juncen Shen, Furong Han, Zhilong Chen, Zhiying Zhang] 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 200bp 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. Springer Basel, 2015 Surrogate reporter nationallicence Single-strand annealing nationallicence Non-homologous end joining nationallicence DNA repair nationallicence Genome editing nationallicence Ren Chonghua College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Xu Kun College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Liu Zhongtian College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Shen Juncen College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Han Furong College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Chen Zhilong College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Zhang Zhiying College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Cellular and Molecular Life Sciences Springer Basel 72/14(2015-07-01), 2763-2772 1420-682X 72:14<2763 2015 72 18 https://doi.org/10.1007/s00018-015-1874-6 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00018-015-1874-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ren Chonghua College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Xu Kun College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Liu Zhongtian College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Shen Juncen College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Han Furong College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Chen Zhilong College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Zhang Zhiying College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 773 0- Cellular and Molecular Life Sciences Springer Basel 72/14(2015-07-01), 2763-2772 1420-682X 72:14<2763 2015 72 18