Ssdna oligonucleotide

Scaffolds containing the two sgRNA target sequences in tandem, inverted, and everted orientations were created using two separate plans. The first plan consisted of a series of overlap extension PCRs on ssDNA oligonucleotides (Integrated DNA Technologies) followed by PCR purification using the MinElute PCR Purification Kit (QIAGEN). The resulting target sequence scaffold oligonucleotides were then subjected to a final amplification with 2× GoTaq Green Master Mix (Promega Corporation) to create poly-dT tails and cloned into the PCR4TOPO vector using the Topo TA Cloning Kit for Sequencing (Invitrogen). The second plan consisted of a series of targeted blunt-end double restriction digests on cloned scaffolds from the first plan, PCR-purification (removing oligonucleotides <∼70 bp) again using the MinElute PCR Purification Kit (QIAGEN), and re-ligation using excess T4 DNA ligase (New England Biolabs). See Supplementary Methods S3 for sequences.

The GO dispersion, cat. No. 763705, of 2 mg/mL concentration in water with mean layer diameter of less than 10 μm and a composition of 42–52% carbon and 44–45% oxygen, was procured from Sigma-Aldrich (St. Louis, MO, USA). The bovine serum albumin (BSA), sodium chloride (NaCl), Trizma^® hydrochloride-99% (Tris-HCl), and sodium dodecyl sulfate (SDS), having the formula CH₃(CH₂)₁₁OSO₃Na, were acquired from Sigma-Aldrich (St. Louis, MO, USA). NC membranes, type 11301, with 8.0 µm pore size and a 47 mm diameter, were purchased from Sartorius (Gottingen, Niedersachsen, Germany). The ssDNA oligonucleotides were acquired from Integrated DNA Technologies, Inc. (Coralville, IA, USA). The used ssDNA oligonucleotide sequences consisted of 5′-TTT CAA CAT CAG TCT GAT AAG CTA TCT CCC-3′ and were labeled at the last primer with 6-fluorescein amidite (6-FAM), forming the complex known as FAM–ssDNA. The αMEM, M4526, and 10% Fetal Bovine Serum, F7524, were bought from Sigma-Aldrich (St. Louis, MO, USA).

Except DNA encoding files from Twist Bioscience, all the DNA oligonucleotides were purchased from Integrated DNA Technologies. Modified oligonucleotides that were purified with high-performance liquid chromatography were purchased, and desalted non-modified oligonucleotides were purchased. Stock solutions (100 and 10 μM) were made using nuclease-free TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5; Integrated DNA Technologies) and stored at −30 °C. DNA encoding for the files was ordered from Twist Bioscience. These files were individually PCR amplified in a 20 µl reaction containing 1 ng DNA pool, 0.5 µM forward and reverse primers, and KAPA HiFi HotStart polymerase. The amplification protocol was as follows: denaturing at 95 °C for 3 min, denaturing at 98 °C for 20 s, annealing at 65 °C for 15 s and extending at 72 °C for 15 s. The second denaturing, annealing and extending steps were repeated 8–10 times, followed by a final extension at 72 °C for 30 s before cooling down to 4 °C. The resulting amplicons were then purified using the Qiagen PCR extraction kit following the manufacturer’s instructions. The files that were obtained this way were then mixed in equal ratios in 10 ng and shipped at room temperature from Seattle to Eindhoven. These templates were then used similar to ssDNA oligonucleotides ordered from Integrated DNA Technologies.