CRISPR-Cas9 Mediated Gene Replacement

For gene replacement based on HDR templates, 5 µg of plasmid DNA templates or 1 µg of ssDNA were used. For tCTS PCR templates, 0.75 µl of each gRNA (2 gRNAs in total) were premixed with 150 µg of poly-L-glutamic acid (PGA, Sigma) as previously described^{29 (link)}. 1.5 µl of TrueCut Cas9 Protein v2 (5 µg/µl, Thermo Fisher Scientific) were then added to the gRNA-PGA complexes and incubated 20 min at room temperature before addition of 750 ng of tCTS PCR template. The different RNP-HDR template complexes were then added to 1×10⁶ cells in buffer R and electroporation was performed using the 10 µl Neon transfection System Kit (ThermoFisher Scientific) using one pulse at 1600V and 30 ms width. When needed, ZsGreen⁺ cells were then sorted using a FACSaria (BD Bioscience).