Table 1.
Putative Safe Harbor Sites for CRISPR Insertion of the Reporter Cassette.
Fig 1.
Map of pCHR1-BmGLuc-GFP: Reporter open reading frames and direction of transcription are indicated by the colored arrows.
Fig 2.
In vitro digestion of the putative insertion site: An amplicon corresponding to Chr1scaffold 001:6484000–6485999 was produced and digested with the corresponding sgRNA and CAS9 nuclease, as described in Materials and Methods. Lane A = undigested amplicon. Lane A+R+C = amplicon incubated with the sgRNA and CAS9 nuclease. Lane A+C = amplicon incubated with CAS9 nuclease alone. Lane A+R = amplicon incubated with the sgRNA alone.
Fig 3.
Gaussia luciferase activity secreted by individual F1 microfilaria: Microfilariae were collected from two gerbils that were independently infected with transfected molting L3 and cultured overnight, and one gerbil infected with untransfected L3. The media for the cultured individual microfilaria were assayed for Gaussia luciferase activity, as described in Materials and Methods. The number of microfilariae producing luciferase activity corresponding to each interval on the X axis are represented by the bars. The Y axis is presented using a logarithmic scale for clarity. #mf = number of mf secreting the indicated range of Gaussia luciferase activity into the culture medium. RLU = Relative light units.
Fig 4.
Hemi-nested PCR analysis of insertion sites in individual microfilaria secreting Gaussia luciferase: Results shown are 12 representatives of those obtained from all 46 luciferase positive microfilariae recovered. Lanes labeled mf+ = DNA prepared from individual microfilaria positive for luciferase activity. Lane mf- = DNA prepared from an individual microfilaria recovered from the animals that did not secrete luciferase. Lane DNA = genomic DNA prepared from untransfected adult female B. malayi DNA.
Fig 5.
Adaptor mediated hemi-nested PCR assay for off target insertion sites: Results shown are representatives from 14 individual transgenic microfilariae and 13 non-transgenic microfilariae, all of which gave results identical to those shown here. Lanes labeled mf- = non-transgenic microfilaria and those labeled mf+ = transgenic microfilaria.