Neon transfection system 10 l kit

Individual sgRNAs targeting either the RFTS, CXXC, BAH1, BAH2, or MTase domains were ordered from IDT (see Supplementary Data 4 for sgRNA sequences). Alt-R CRISPR-Cas9 tracrRNA (1072533) and Alt-R S.p. Cas9 nuclease (1081059) were also obtained by IDT. CRISPR Cas9 RNP transfections were performed according to IDT’s protocol using the Neon transfection system 10 µL Kit (Thermo Fisher Scientific). Cells were pelleted for 5 min, 300 × g 48 h post-transfection, and resuspended in 50 µL DirectPCR Cell Lysis Buffer (Viagen Biotech) containing 2 µL Proteinase K (Thermo Fisher Scientific). Lysates were incubated at 68 °C for 15 min, followed by 10 min at 98 °C. Regions of DNMT1 were PCR amplified and purified using the QIAquick PCR Purification Kit using the manufacturer’s instructions (Qiagen). Purified PCR product was sent to Genewiz (South Plainfield, NJ) for Amplicon-EZ Next Generation Sequencing and analyses. The following primers were used for PCR amplification: RFTS-F 5′-GAGCAGCTGTAGGCCAAGTC-3′; RFTS-R 5′-AGGCTACCCCAACTGAACCT-3′; CXXC-F 5′-TGGTGGTGTGATCTTGGCTA-3′; CXXC-R 5′-CTGACCTACCTCCGCTCTTG-3′; BAH1-F 5′-GTGGGGGACTGTGTCTCTGT-3′; BAH1-R 5′-TGAAAGCTGCATGTCCTCAC-3′; BAH2-F 5′-GGGGGAGTCTACCTTGCAGT-3′; BAH2-R 5′-CGAGGAAGTAGAAGCGGTTG-3′; SAM-H-F 5′-GTTGCAGTGAGCCAAGATCA-3′; SAM-H-R 5′-TGACGGTTGTGCTGAAGAAG-3′; SAM-L-H 5′-TCACTTGAGCCTCTGGGTCT-3′; SAM-L-R 5′-ATCAGTGCATGTTGGGGATT-3′.

ScreenFect A-Plus (Incella GmbH, Eggenstein-Leopoldshafen, Germany) was used to transfect HEK293T cells following the manufacturer’s instructions. For MCPyV⁺ cells, the 24-well optimization protocol of the Neon™ Transfection System 10 µL Kit (Thermo Fisher Scientific, Merelbeke, Belgium) was applied. Briefly, 2 × 10⁵ cells/condition were electroporated with 1 µg of DNA and plated in 24-well plates. Transfection efficiency was quantified by flow cytometry (BD Accuri C6, BD Biosciences, San Jose, CA, USA). Once the optimal conditions were selected, 10 µg of DNA was used to electroporate 3–5 × 10⁶ cells for further analysis using the 100 µL kit.

Rett syndrome PDFs were obtained from the Rett Syndrome Research Trust and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Genesee Scientific #25-500) supplemented with 15% fetal bovine serum (Gibco #26140079) and 1x nonessential amino acids (Gibco #11140050). These cells were also incubated at 37 °C with 5% CO₂. PDF electroporation’s were performed using the Neon Transfection System 10 µl kit (ThermoFisher #MPK1096) as previously described^{17 (link)}. A total of 500 ng ABE mRNA and 100 pmol sgRNA were electroporated into ~50,000 PDF cells. 48 h post-electroporation, genomic DNA was extracted with QuickExtract (Lucigen #QE09050) for amplicon sequencing.