Table 1 Optimization of Cas9, crRNA, and rescue template concentrations
Experiment numberExperimentInsertion in gtbp-1 (size)Cas9 pmol/┬ÁlCas9 sourcecrRNA ratio (gtbp-1/dpy-10)template ratio (gtbp-1/ dpy-10)Percent P0 with rollers (n)Percent edits rollers (n)Percent edits nonrollers (n)
AP80BaselinemCherry (700 bp)20.6Homemade1 (15.7/15.7)1.3 (0.56/0.43)80% (15)24.4% (131)2.9% (134)
AP78Increase crRNA ratiomCherry (700 bp)20.6Homemade2.5 (39.4/15.7)1.3 (0.56/0.43)50% (20)51.9% (131)17.8% (174)
AP111Decrease Cas9 concentrationmCherry (700 bp)4.1Homemade2.5 (39.4/15.7)1.3 (0.57/0.43)33% (18)33.3% (12)12.7% (212)
AP96Increase template ratiomCherry (700 bp)15.5Homemade2.5 (29.6/11.8)2.5 (1.13/0.44)45% (11)70.2% (84)22.1% (122)
AP108Lower crRNA concentrationsmCherry (700 bp)15.5Homemade2.5 (11.1/4.4)2.5 (1.13/0.44)10% (10)60% (10)10% (40)
AP112Plasmid deliverymCherry (700 bp)NAPlasmid2.5 (0.025/0.01)1.3 (0.56/0.43)11% (17)83% (18)14% (120)
AP60Commercial Cas9eGFP (800 bp)4.1Commercial4.2 (39.4/9.4)0.9 (0.40/0.43)50% (30)55% (89)18.9% (37)
AP58Commercial Cas9 + Increase template ratioeGFP (800 bp)4.1Commercial4.2 (39.4/9.4)2.1 (0.91/0.43)61.5% (25)61.1% (54)52.1% (46)
  • All experiments were conducted as described in Figure 1 by cotargeting dpy-10 and gtbp-1. Concentrations are in picomoles per microliter. Percent P0 with rollers, percent of injected hermaphrodites that segregated rollers (n= total number of injected hermaphrodites); Percent edits rollers, percent FP+ edits at the gtbp-1 locus among rollers from the top three broods with the most rollers (n= number of rollers screened for FP expression); Percent edits nonrollers, percent FP+ edits at the gtbp-1 locus among nonrollers from the top three broods with the most rollers (n= number of nonrollers screened for FP expression).