Dual-reporter surrogate systems for efficient enrichment of genetically modified cells.
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.
College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Chonghua
C
Ren
Kun
K
Xu
Zhongtian
Z
Liu
Juncen
J
Shen
Furong
F
Han
Zhilong
Z
Chen
Zhiying
Z
Zhang
D002469:Cell Separation / Q000379:methods*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D005818:Genetic Engineering; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D008957:Models, Genetic