Rame-shifting mutations generated at the target sequences can make GFP and H-2Kk in frame, leading to the expression of GFP and H-2Kk. To test this reporter system, we cotransfected plasmids encoding the CCR5-specific ZFN (Z891) [23] and its reporter into HEK293 cells. CCR5 is a coreceptor of human immunodeficiency virus (HIV) and the knockout of this gene using ZFNs has been reported to prevent HIV infection into T cells [25,26]. One day after transfection, a significant fraction of cells expressed mRFP, whereas eGFPexpressing cells were hardly observed (Figure S1). The number of eGFP-expressing cells gradually increased over 3 days, suggesting that the ZFN cleaved the target sequence in the reporter plasmid to induce frame-shifting indels [3]. Three days after transfection, H-2Kk-expressing cells were magnetically separated after labeling with anti-H-2Kk antibody conjugated with magnetic beads. Fluorescent microscopy showed that magnetically separated cells were enriched with GFP+ cells (Figure 2A). We measured the mutation frequencies (or indel ) in sorted and unsorted cells using T7 endonuclease I (T7E1), an enzyme that specifically recognizes and cleaves heteroduplexes formed by the hybridization of wild-type DNA sequences and IKK 16 site mutant sequences. This assay showed that the mutation Sapanisertib frequency at the CCR5 gene in H2Kk+ cells was 46 , 12-fold higher than that in unseparated cells (3.7 ) (Figure 2B), demonstrating efficient enrichment of CCR5disrupted cells. To confirm this strong enrichment of mutant cells, we next determined the DNA sequences around the target site, and found that the mutation frequency in the magnetically separated cells was 60 , 21-fold higher than that in unseparated cells (Figure 2C). The relatively lower fold enrichment observed with the T7E1 assay as compared to DNA sequencing may be attributable to the fact that at high mutation frequencies, mutant sequences can form homoduplexes, which are insensitive to digestion by T7E1. Thus, the T7E1 assay often underestimates fold enrichments [3]. Next, we tested whether this reporter system is portable to other ZFNs and TALENs. For this, we first used this reporter system with a TP53 gene-targeting ZFN pair [3] in HEK293 cells. TP53targeting ZFNs can be used to mutate or repair TP53, an important tumor suppressor gene [27]. The T7E1 assay showed that the mutation frequency in magnetically separated cells was 25 , 17-fold higher than that in unseparated cells (1.5 ) (Figure 3A). We next tested this reporter using a CD81-targeting ZFN pair in a different cell line, Huh 7.5 cells (a human hepatocyte cell line). The T7E1 assay revealed that the mutation frequency 1317923 was 8.6 , whereas that in the unseparated group was below the detection range (,0.5 ) (Figure 3B), suggesting at least 17-fold enrichment of mutant cells. When we performed this reporter-mediated magnetic separation using a BRCA1-targeting TALEN pair, the T7E1 assay showed that the mutation frequency in the H-2Kk+ cells was 47 , 17-fold higher than that in unseparated cells (2.7 ) (Figure 3C), suggesting that this magnetic reporter system is compatible with TALENs as well.Flow Cytometer-Free Enrichment of Mutant CellsFigure 4. Overview of the episomal reporters used for the enrichment of nuclease-induced mutant cells via hygromycin selection. (A) The working mechanism of the hygromycin reporter. mRFP is constitutively expressed by the CMV promoter (PCMV), whereas the HygroR-eGFP fusion gene is not expressed in the a.Rame-shifting mutations generated at the target sequences can make GFP and H-2Kk in frame, leading to the expression of GFP and H-2Kk. To test this reporter system, we cotransfected plasmids encoding the CCR5-specific ZFN (Z891) [23] and its reporter into HEK293 cells. CCR5 is a coreceptor of human immunodeficiency virus (HIV) and the knockout of this gene using ZFNs has been reported to prevent HIV infection into T cells [25,26]. One day after transfection, a significant fraction of cells expressed mRFP, whereas eGFPexpressing cells were hardly observed (Figure S1). The number of eGFP-expressing cells gradually increased over 3 days, suggesting that the ZFN cleaved the target sequence in the reporter plasmid to induce frame-shifting indels [3]. Three days after transfection, H-2Kk-expressing cells were magnetically separated after labeling with anti-H-2Kk antibody conjugated with magnetic beads. Fluorescent microscopy showed that magnetically separated cells were enriched with GFP+ cells (Figure 2A). We measured the mutation frequencies (or indel ) in sorted and unsorted cells using T7 endonuclease I (T7E1), an enzyme that specifically recognizes and cleaves heteroduplexes formed by the hybridization of wild-type DNA sequences and mutant sequences. This assay showed that the mutation frequency at the CCR5 gene in H2Kk+ cells was 46 , 12-fold higher than that in unseparated cells (3.7 ) (Figure 2B), demonstrating efficient enrichment of CCR5disrupted cells. To confirm this strong enrichment of mutant cells, we next determined the DNA sequences around the target site, and found that the mutation frequency in the magnetically separated cells was 60 , 21-fold higher than that in unseparated cells (Figure 2C). The relatively lower fold enrichment observed with the T7E1 assay as compared to DNA sequencing may be attributable to the fact that at high mutation frequencies, mutant sequences can form homoduplexes, which are insensitive to digestion by T7E1. Thus, the T7E1 assay often underestimates fold enrichments [3]. Next, we tested whether this reporter system is portable to other ZFNs and TALENs. For this, we first used this reporter system with a TP53 gene-targeting ZFN pair [3] in HEK293 cells. TP53targeting ZFNs can be used to mutate or repair TP53, an important tumor suppressor gene [27]. The T7E1 assay showed that the mutation frequency in magnetically separated cells was 25 , 17-fold higher than that in unseparated cells (1.5 ) (Figure 3A). We next tested this reporter using a CD81-targeting ZFN pair in a different cell line, Huh 7.5 cells (a human hepatocyte cell line). The T7E1 assay revealed that the mutation frequency 1317923 was 8.6 , whereas that in the unseparated group was below the detection range (,0.5 ) (Figure 3B), suggesting at least 17-fold enrichment of mutant cells. When we performed this reporter-mediated magnetic separation using a BRCA1-targeting TALEN pair, the T7E1 assay showed that the mutation frequency in the H-2Kk+ cells was 47 , 17-fold higher than that in unseparated cells (2.7 ) (Figure 3C), suggesting that this magnetic reporter system is compatible with TALENs as well.Flow Cytometer-Free Enrichment of Mutant CellsFigure 4. Overview of the episomal reporters used for the enrichment of nuclease-induced mutant cells via hygromycin selection. (A) The working mechanism of the hygromycin reporter. mRFP is constitutively expressed by the CMV promoter (PCMV), whereas the HygroR-eGFP fusion gene is not expressed in the a.
