Life Technologies

55 Life Technologies ™ | Genome editing with enriching capabilities Engineered nuclease recognition sequences Stop + Engineered nuclease Double-strand break repair Restore OFP and CD4 expression OF = N terminus of the OFP gene FP = C terminus of the OFP gene V5 PCMV OF FP T2A T2A T2A CD4 Stop V5 PCMV V5 V5 OF V5 PCMV OFP CD4 FP CD4 T2A FP CD4 Figure 1 ê ê ê X X Stop V5 PCMV V5 OF X Engineered nuclease recognition sequences Stop + Engineered nuclease Double-strand break repair Restore OFP and CD4 expression OF = N terminus of the OFP gene FP = C terminus of the OFP gene V5 PCMV OF FP T2A T2A T2A CD4 Stop V5 PCMV V5 V5 OF V5 PCMV OFP CD4 FP CD4 T2A FP CD4 Figure 1 ê ê ê X X Stop V5 PCMV V5 OF X Figure 15. Simple, rapid evaluation of the functionality of the programmable nuclease and direct enrichment of the genome-modified cells. The GeneArt ® Genomic Cleavage Selection Vector contains an orange fluorescent protein (OFP) reporter gene for quickly detecting the functionality of the engineered nucleases in the transfected cells. The OFP and CD4 reporters can be used for enrichment of the nuclease-modified cells using fluorescence-activated cell sorting (FACS) or CD4 antibody–conjugated Dynabeads ® particles. The vector has been constructed such that the N-terminal and C-terminal ends of the OFP gene are separated by a cloning site for the target sequence of the programmable nuclease. The upstream sequence coding for the N-terminal portion of the OFP gene contains a region complementary to the 5´ end of the C-terminal region of the OFP gene. Three stop codons are inserted following the upstream coding sequence of the N-terminal OFP sequence to ensure no expression of the reporter OFP prior to nuclease activity. The CD4 gene is out of frame for expression when the OFP gene is interrupted by the cloning site. When a double-strand break is introduced into the target sequence by the programmable nuclease, the complementary strands from each end sequence of OFP will recombine to restore OFP expression, and the CD4 gene is now in frame for expression. Thus, cleavage by TAL, CRISPR, or zinc finger nucleases can be checked as early as 24 hours posttransfection by simply viewing the transfected cells under a fluorescence microscope. The percentage of OFP-positive cells will reflect the cleavage activity of the TAL, CRISPR, or zinc finger nuclease. The OFP-positive cells can be enriched through FACS. Additionally, the membrane protein CD4 coding gene is fused to OFP through T2A self-cleavage peptide, so the nuclease-modified cells can be enriched using Dynabeads ® particles conjugated with anti-CD4 antibody.

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