Chromosome-specific CRISPR-Cas9 Nucleofection

Guide RNAs (gRNAs) recognizing chromosomes 1p and 19q sequences were designed using the CRISPick website (https://portals.broadinstitute.org/gppx/crispick/public). Two gRNAs for each chromosome arm were chemically synthesized (Synthego) (Supplementary Table S1). Nucleofection of ribonucleoprotein (RNP) complexes consisting of gRNAs and Cas9 was performed as described with modifications.^{19 (link)} In brief, 400 pmol of HiFi Cas9 (Integrated DNA Technologies) and 500 pmol sgRNA were mixed and incubated at room temperature for 10 minutes. 1 × 10⁶ cells were washed with Opti-MEM (ThermoFisher Scientific) and resuspended in 100 μl Nucleofector Solution (Lonza), gently mixed with pre-assembled RNPs, and electroporated with appropriate programs using Nucleofector 2b or 4d (Lonza). The nucleofection program used for HEK 293T, LN-229, and U-251 cells was Q-001, X-009, and DS-138, respectively.