Fig. 3.
Parameters affecting cassette excision by MMEJ. a Schematic of the plasmid-based MMEJ assay measuring luciferase repair. b Luciferase activity as a function of increasing flanking μH length. Inset shows luciferase activity with 5 bp μH compared to background (0 bp). Error bars show s.e.m. (n = 3). c Schematic of the HPRT1 chromosomal assay depicting MhAX cassettes and nested ps1 protospacers with flanking 11 or 29 bp of μH. Synonymous mutations are shown in red. d HATR colonies arising from targeted clones without or with nuclease (pX-ps1) transfection. One representative clone is shown for each homology length. e Schematic of HPRT1-targeted μ29 MhAX cassettes with inverted ps1 protospacers. Predicted heterology lengths are indicated for each DSB. HAT-resistant colonies following excision are representative of three independent experiments. HAT-selected populations from either protospacer orientation are enriched for MMEJ repair (Supplementary Fig. 12c)