Extended Data Fig. 1. Micronucleus formation after CRISPR-Cas9 genome editing in several cell lines.

(a) Experimental schemes. Top, RNP transfection. Bottom, inducible Cas9 expression with constitutive expression of gRNAs (RPE-1 cells). G₀ cell cycle block was by serum starvation. Dividing cell cartoon represents approximate time of cell division.

(b) Micronucleation frequency after CRISPR-Cas9 RNP transfection in asynchronous cells. Left, editing efficiency. Right, frequency of micronucleation for these RNP transfections. (n = 3 experiments with 1339, 1231, 1220, 1236, and 1237 cells scored, left to right). Error bars: mean +/− SEM, two-tailed Fisher’s exact test.

(c) Representative Western blot of Cas9 levels at the indicated times after induction with doxycycline. 1^st division is 24 hours after serum starve release, and 2^nd division is 48 hours after release. Dox is doxycycline. n = 3 experiments.

(d) Number of cleaved chromosome arms contained within micronuclei for the indicated gRNAs and Cas9 expression strategies (RPE-1 cells) determined by FISH to detect the centromere (RNP Cas9) and/or subtelomere of the targeted chromosome (RNP Cas9 and Dox-inducible Cas9). RNP Cas9: for 2p: n = 2 experiments with 64 micronuclei counted, 4q: n = 2 experiments with 58 micronuclei counted, 5q: n= 3 experiments with 116 micronuclei counted, Xq: n = 2 experiments with 96 micronuclei counted; (Dox) Doxycycline-inducible Cas9; n = 3 experiments; 168 micronuclei counted per condition.

(e) Frequency of micronucleation in synchronized BJ fibroblasts after RNP transfection; (n = 3 experiments with 2378, 2487, 2423, 2714 cells, left to right). Error bars: mean +/− SEM, two-tailed Fisher’s exact test.

(f) Left, percentage of MN containing the targeted chromosome arm for the chr5q-targeting gRNA in BJ cells, as counted using subtelomeric FISH probes. Right, the number of chr5q chromosome arms per micronucleus in BJ cells, determined from centromere-specific and subtelomere-specific FISH probes. (n = 2 experiments counting 109 micronuclei).

(g) Cut site and FISH probe locations for allele-specific gRNA experiments. PAM sequence is in bold, with the polymorphic site in red. Orange star is the centromere FISH probe and green circle the subtelomere FISH probe. gRNAs target the reference allele.

(h) Editing efficiency after Cas9/gRNA RNP transfection with allele-specific gRNAs. (n = 3 experiments). Error bars: mean +/− SEM.

(i) Micronucleation frequency from samples in (h). (n = 3 experiments with 7066, 7041, 7253, cells scored for micronucleation, left to right). Error bars: mean +/− SEM, two-tailed Fisher’s exact test.

(j) Left, percentage of MN containing the targeted chromosome arm for the allele-specific gRNAs, as scored using subtelomeric FISH probes. Right, pie chart of the number of targeted arms per micronucleus in RPE-1 cells, as determined from subtelomere-specific FISH probes. (n = 3 experiments counting 123 and 184 micronuclei, left to right) Error bars: mean +/− SEM.