Forward and reverse adaptors

In total, 1 × 10⁷ A549 cells were used per 4C assay. Cells are treated with formaldehyde, which cross-links proteins to proteins and DNA. Cross-linked chromatin is subsequently digested with HindIII (NEB). Next, chromatin is diluted and then religated by T4 ligase (NEB) to fuse the ends of DNA fragments. DNA is further digested by DpnII (NEB) that digests the fragment into smaller fragments after the removal of cross-links by heating. These fragments are religated under diluted conditions to create much smaller DNA circles using T4 ligase (NEB) again. Inverse PCR primers with Illumina forward and reverse adaptors were designed to anneal to a bait locus HindIII/DpnII restriction fragment. A total of 3200 ng of 4C template was used to amplify each bait using Expand Long Template Polymerase (Roche) system. The PCR program is as follows: 2 min at 94 °C; 10 s at 94 °C; 1 min at bait specific annealing temperature; 3 min at 68 °C; 29 × repeat; 5 min at 68 °C; hold at 4 °C. All 16 PCR tubes were pooled and purified using High Pure PCR Product Purification Kit (Roche) kit. Subsequently, high-throughput sequencing is used to detect the captured regions.

The CRISPOR program (http://crispor.tefor.net) (68 (link)) computational tool was used to predict gRNA-dependent off-target editing sites for each mutation (Supplemental Figure S2). The on-target guide RNA associated with each mutation was set as the query sequence with an NGN PAM. From the list generated from CRISPOR, we selected the top 10 predicted off-target sites ranked by cutting frequency determination (CFD) score in descending order. Primers were designed using the IDT primer design software (IDT, Coralville, IA) with the Illumina forward and reverse adaptors as mentioned (see Table 3). Amplified genomic DNA off-target sites were sequenced using next generation sequencing.