FIG 1.

Generation of persistent, targeted breaks in G₁ phase cells using CRISPR/Cas9. (A) Work flow for CRISPR/Cas9-induced breaks in LigIV^−/−:iCas9 v-Abl-transformed pre-B cells. (B) Intracellular staining and flow cytometric analysis for FLAG-Cas9 in cycling (top) and G₁-arrested (bottom) LigIV^−/−:iCas9 cells that were untreated or treated with doxycycline for the indicated times. (C) Staining for the Thy1.1 cell surface marker and flow cytometric analysis for Thy1.1 in G₁-arrested LigIV^−/−:iCas9 cells nucleofected with the pKLV-U6gRNA(BbsI)-UbcThy1.1 gRNA expression vector and nonnucleofected cells. (D) (Top) Schematic of the Southern blotting strategy to detect cleaved alleles at the enhancer of Tcrb (Eb). (Bottom) Southern blot showing intact and cut Eb alleles at 0 and 24 h after nucleofection of LigIV^−/−:iCas9 cells with a gRNA plasmid targeting Eb (gEb). (E) UCSC Genome Browser screenshot depicting the γ-H2AX ChIP-seq signal at the Eb locus in G₁-arrested, LigIV^−/−:iCas9 cells at 24 h after nucleofection with gEb (bottom track) or no gRNA (top track). The arrow indicates the gEb target site. Dox and dox, doxycycline; SSC, side scatter; FSC, forward scatter.