Dna oligo

PCR was performed using Q5® High-Fidelity DNA Polymerase (M0491, NEB) with DNA oligos from Integrated DNA Technologies (IDT). PCR products were gel purified using Gel Purification Kit (D4002, Zymo Research). To generate entry vectors, DNA fragments were inserted into BsaI-digested GreenGate empty entry vectors [52 (link)] via Gibson assembly (2× NEBuilder Hifi DNA Assembly Mix, NEB) or restriction ligation with T4 DNA ligase (NEB). Base editors, gRNAs, and fluorescent reporter vectors were assembled using Golden Gate cloning (30 cycles (37°C, 5 min; 16°C, 5 min); 50°C for 5 min; 80°C for 5 min) with BsaI or BbsI [52 (link)]. Vectors were transformed by heat-shock transformation into DH5α E.coli or One Shot™ ccdB Survival™ competent cells (Thermo Fisher Scientific). Cells were plated on lysogeny broth medium containing 100 μg mL^-1 carbenicillin, 100 μg mL⁻¹ spectinomycin, 25 μg mL⁻¹ kanamycin, or 40 μg mL⁻¹ gentamycin, depending on the selectable marker. Plasmids were isolated (GeneJET Plasmid Miniprep kit, Thermo Fisher Scientific) and confirmed by restriction enzyme digestion and/or Sanger sequencing (Eurofins, Mix2seq). All plasmids are described in Additional file 3. All primers are listed in Additional file 4.

All chemicals were purchased from Fisher Scientific (Hampton, NH, USA), Sigma-Aldrich (St. Louis, MO, USA), Thomas Scientific (Swaysboro, NJ, USA) or VWR (Radnor, PA, USA). DNA oligos were purchased from Integrated DNA Technologies (IDT) (Coralville, IA, USA) or Eurofins Genomics. Restriction enzymes were purchased from Thermo Scientific (Waltham, MA, USA). DNA sequences were analyzed by Eurofins Genomics (Louisville, KY, USA). pcDNA3.1-mMaroon1, pcDNA3-mCarmine were gifts from Michael Lin (Addgene plasmid #83840) and Oliver Griesbeck (Addgene plasmid #109486), respectively. Absorbance and fluorescence measurements were performed with a monochromator-based BioTek Synergy Mx Microplate Reader.

DNA oligos, cholesterol-modified
ssDNA, amine-modified ssDNA, and biotin-modified ssDNA (Tables S1–S4) were purchased from Integrated
DNA Technologies (Coralville, IA). DBCO-Cy5 and DBCO-Sulfo-NHS-Ester,
Sodium Chloride and Paraformaldehyde, and PEG 8000 were purchased
from Sigma-Aldrich. Streptavidin-Alexa Fluor 488 and 647 conjugates
were purchased from Invitrogen. Roswell Park Memorial Institute (RPMI)
1640 Medium, Dulbecco’s Phosphate-Buffered Saline (PBS) was
purchased from Corning. AFM tips were purchased form NanoAndMore (OMCL-AC160TS).
ReadyProbes Cell viability imaging kit (Blue/Green) and SYBR Safe
DNA gel stain were purchased from Invitrogen. HUVECs, Endothelial
Cell Growth Medium-2 BulletKit was purchased from Lonza.