Cas9 RNP transfection in immortalized cell lines

sgRNAs were synthesized with the Trueguide Synthetic gRNA platform (Thermo Fisher Scientific) as chemically modified custom oligos, where the final 3 bases on both the 5’ and 3’ end of the sgRNA are 2’-O-Methyl bases and the linkages between them are phosphorothioates, in order to increase editing efficiency and protect from nuclease degradation. Their sequences are listed in Supplementary Table 1.

RNP complexes were prepared following a modified version of the suggested manufacturer’s protocol. Briefly, gRNA/Cas9 complexes were formed by incubating 250 ng of the gRNA with 1 μg of purified Cas9 protein (TrueCut Cas9 Protein v2, Invitrogen) in OptiMEM (Invitrogen). Cells were seeded on 12-well dishes, #1.5 glass coverslips (fixed imaging experiments), or 35-mm gridded ibiTreat dishes (ibidi) (Look-Seq), were synchronized by serum starvation in 0.1 % FBS-containing media for 24 h where applicable, and subjected to Cas9 RNP transfection 22 h upon release from the block. Transfection of ribonucleoprotein complexes was performed using Lipofectamine CRISPRMAX Reagent (Invitrogen). Cells were fixed 46 h after release from block to measure the percentage of cells with micronuclei and 35 – 40 h after release for the FISH experiments.