Hcas9 d10a

For generating the dCas9-eGFP construct, plasmid hCas9_D10A was purchased from Addgene (addgene ID: 41816²⁴ (link)). Inactivation of the second nuclease domain (H840A) was performed by site-directed mutagenesis using primers dCas1-F, dCas2-R, dCas2-F, and dCas3-R. The resulting PCR-product (dCas9) was digested with BsrGI and XbaI and ligated into pCAG.⁶⁹ (link)^,70 (link) The NLS-eGFP-sequence was amplified by PCR using primers eGFP1 and eGFP2, digested with AsiSI and NotI and ligated downstream of dCas9. pEX-A-U6-gRNA was synthesized at Eurofins MWG Operon according to Mali et al.²⁴ (link) gRNA-expression vectors were generated by amplifying pEX-A-U6-gRNA with forward and reverse primers, which introduced the protospacer sequence for minor satellites repeats (MiS), major satellites repeats (MaS) and telomeres (Tel), respectively.
Nucleotide sequences:

PER.C6 cells were plated at a density of 1.5 × 10⁶ cells per well of 6-well plates (Greiner Bio-One). The next day, a total amount of 6 μg of DNA corresponding to 1:1 mixtures of hCas9 (Addgene plasmid 41815) and gRNA_AAVS1-T2 (Addgene plasmid 41818), pAdSh.PGK.Cas9 and pAdSh.U6.gRNA^S1, hCas9 and gRNA_Cloning Vector (Addgene plasmid 41824) or hCas9_D10A (Addgene plasmid 41816) and gRNA_AAVS1-T2, were transfected by deploying a 1 mg/ml polyethyleneimine (PEI) solution (Polysciences) essentially as described elsewhere41 (link) except for the use of 6 μg of DNA and 19.7 μl of PEI instead of 6.25 μg of DNA and 18.8 μl of PEI. At 3 days post-transfection genomic DNA from mock-transfected cells and from co-transfected cells, was isolated according to a previously described method42 (link). Targeted gene disruption was assessed by using the T7EI-based assay as described below.