Stable derivatives of SVG-A cells expressing Cas9 and gRNAs show no InDel mutations in off-target genes.

<p>The SURVEYOR assay was used to detect the presence of mutations in PCR products derived from SVG-A cells expressing Cas9 and gRNAs. <i>m</i> 1 (A), m2 (B) and m3 (C). Human cellular genes with the highest degree of homology to each motif were identified by BLAST search at the NCBI website (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>). For each motif, PCR product was amplified from the top three genes with the highest degree of homology and examined for InDel mutations using the SURVEYOR assay as described in Materials and Methods. Amplification of T-Ag was the positive control in each panel. <b>A.</b> For motif m1, we amplified M12 (NM_017821, human RHBDL2 rhomboid, veinlet-like 2 (Drosophila), Gene ID: 54933, NCBI Ref Seq: NC_000001.11, >gi|568815597:c38941830-38885806), M17 (NM_001243540, human KIAA1731NL, Gene ID: 100653515, NCBI Ref Seq: NC_000017.11, >gi|568815581:78887721–78903217) and M19 (NM_016252, human BIRC6 baculoviral IAP repeat containing 6, Gene ID: 57448, NCBI Ref Seq: NC_000002.12); <b>B.</b> For motif m2, we amplified M21 (NM_012090, human MACF1 microtubule-actin crosslinking factor 1, Gene ID: 23499, NCBI Ref Seq: NC_000001.11, >gi|568815597:39084167–39487138), M23 (NM_005898, human CAPRIN1 cell cycle associated protein 1, Gene ID: 4076, NCBI Ref Seq: NC_000011.10, >gi|568815587:34051683–34102610), M24 (NM_024562, human TANGO6 transport and Golgi organization 6 homolog (Drosophila), Gene ID: 79613, NCBI Ref Seq: NC_000016.10, >gi|568815582:68843553–69085482); <b>C.</b> For motif m3, we amplified M31 (NM_001048194, human RCC1 regulator of chromosome condensation 1, Gene ID: 1104, NCBI Ref Seq: NC_000001.11, >gi|568815597:28505943–28539196), M32 (NM_004673, human ANGPTL1 angiopoietin-like 1, Gene ID: 9068, NCBI Ref Seq: NC_000001.11, >gi|568815597:c178871353-178849535), M33 (NM_174944, human TSSK4 testis-specific serine kinase 4, Gene ID: 283629, NCBI Ref Seq: NC_000014.9, >gi|568815584:24205530–24208248).