Chapter 4 —— 84 —— was then subjected to Illumina MiSeq next-generation amplicon deep sequencing for obtaining 50000 paired-end reads covering the aforementioned genomic regions. The pipeline and reagents used for this amplicon deep sequencing analysis have been previously specified in detail [25,35,36]. Gene-editing specificity assays EGFP-expressing hMSCs generated via co-transduction with AAV-HRS1 donor and adenovectors encoding high-specificity eCas94NLS:GS1 or regular Cas9:GS1 complexes, were sorted through a CytoFLEX SRT Cell Sorter (Beckman) and, subsequently, were subjected to genomic DNA extraction by using the DNeasy Blood & Tissue Kit according to the manufacturer’s instructions. The AAV vector DNA insertions at on-target AAVS1 and at offtarget CPNE5 sequences were captured by junction PCR assays with the aid of Platinum SuperFi II DNA Polymerase (Thermo Fisher Scientific; cat. no.: 12361010). Amplicons specific for EGFP served as internal controls. The PCR primers and cycling conditions used in these junction PCR assays are listed in Supplementary Tables S6 – S9, respectively. EGFP-expressing HeLa cells generated by combining transfections of plasmids encoding Cas9:GS1, eCas94NLS:GS1 or eCas9.D10A4NLS:GS1 with transductions with the AAV-HRS1 donor (1.0 TU cell-1), were sorted and subjected to genomic DNA isolation by using the DNeasy Blood & Tissue Kit following the manufacturer’s instructions. The AAV-HRS1 transductions took place at 6 h post-transfection with the transfections having been performed with the reagents and conditions indicated in Supplementary Table S10 on 1 × 105 HeLa cells seeded one day before in wells of 24-well plates. Prior to EGFP-directed cell sorting and genomic DNA isolation, the transfected and transduced HeLa cells were sub-cultured for 20 days. Finally, assessing AAV donor DNA insertions at the CPNE5 off-target site was conducted through PCR with the primers #2142 and #2145 with sequences 5′-CCTTGGATTCCTCATCCCAG-3′) and 5′-CCCAGGGCTCTACTCAC
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