Fig. 4
Impact of the CRISPY mix on gene fragment insertion efficiency in iCRISPR hiPSCs. Shown is the insertion efficiency of a gene fragment coding for a blue fluorescent protein (BFP) in the heterozygous AAVS1 iCRISPR locus using a single-stranded DNA donor in 409-B2 iCRISPR-Cas9n hiPSCs. The design of the mtagBFP230 ssODN donor and the iCRISPR system is shown in a. We inserted a 871 nt (including 50 nt homology arms) sequence coding for a 2A-self cleaving peptide in front of a blue fluorescent protein (BFP), directly after the N-terminal nuclear localization signal sequence (NLS) of the Cas9n in the heterozygous AAVS1 iCRISPR locus13. If the sequence is inserted, doxycycline will lead to expression of nucleus-imported BFP. Representative images of the mock, NU7026, and CRISPY mix treatment, after 7 days of BFP expression are shown in b as phase contrast (PC), propidium iodide nuclei staining (PI), mtagBFP2 expression (BFP), and merge of PI and BFP. Two images (× 50 magnification, white size bar 200 µm) from each of three technical replicates for the respective treatments were used to quantify the percentage of cells with BFP insertion using ImageJ (c)
