Fig 1: PM and/or DT selection of MCF7 cells transfected with DPH gene-specific CRISPR/Cas9 expression constructs and pac donor plasmids. Mean values + SEM are shown (n = 4, ***p < 0.001). (A) PM selection generates resistant colonies at a 2-fold higher frequency when DPH1 gRNA is used compared with scRNA. Combining PM selection and DT selection reveals the frequency at which the pac cassette becomes integrated in cells in which both DPH1 alleles are inactivated. DPH1 gRNA generates clones with PM-DT double resistance. scRNA generates only PMr colonies and no DTr colonies. (B) Comparison of the frequency of DTr (both DPH1 genes inactivated) colonies and PMr (pac integration at DPH1 or at another site) colonies. The position or zygosity of pac integration cannot be determined. (C) MCF7 cells transfected with DPH2-specific gRNA and donor DNA were subjected to PM and/or to DT selection. The absolute numbers of gRNA- as well as scRNA-mediated editing events are reduced for DPH2 compared with DPH1. The efficacy of targeted inactivation and integration may be due to differences in the sequence of the gRNA and homology arms and/or target gene accessibility. Reduced ‘efficacy’ of scRNA-mediated integration is a consequence of sequence features within the different homology arms of the pac cassette, as the scRNA was identical in the DPH1 and DPH2 editing experiments.
Supplier Page from OriGene Technologies for DPH2 Human Gene Knockout Kit (CRISPR)