Loading dye

A Zymoclean Gel RNA Recovery Kit from Zymo Research was used to purify ssDNA from agarose gels. The gel bands containing target ssDNA were excised with a clean razor blade. Three times the gel slice volume of the provided agarose dissolving/binding buffer was added to each excised gel band and melted at 55 °C on a heat block for 15 min. Each dissolved gel solution was transferred to a provided silica-based spin column and centrifuged at 12,000 rcf for 2 min. 400 µL RNA Prep buffer was added to each spin column followed by centrifugation at 12,000 rcf for 1 min. Washing was carried out by the addition of 800 µL ethanol-based wash buffer followed by centrifugation at 12,000 rcf for 30 s. After repeating the washing step with 400 µL ethanol-based wash buffer, each spin column was centrifuged at 12,000 rcf for 2 min to remove residual ethanol. Flow-through in all steps was discarded. After transferring each spin column to clean microcentrifuge tubes, 6–20 µL of provided nuclease-free water was added directly to the column matrix, and the spin columns were centrifuged at 10,000 rcf for 1 min for retentate collection. A fraction of each purified ssDNA was mixed with 6 × loading dye (NEB) and the yield was estimated by gel electrophoresis as described above^{24 (link)}.

DNA samples and 1 kilobases plus DNA marker were mixed with 6 × loading dye (New England BioLabs, USA), loaded onto a 1% agarose gel in Tris–acetate-EDTA buffer and run at 110 V for 50 min. RNA samples were run and analysed using the FlashGel RNA cassette system (Lonza Bioscience, USA).
Additionally, saRNA samples were prepared according to the Agilent RNA 6000 Nano Kit guide and run on the Agilent Bioanalyzer 2100 using the mRNA Nano assay class protocol. The data was analysed by the 2100 Expert software (B.02.10.S1764). Fraction of intact saRNA based on the electrogram was represented by area under the curve (Supplementary information). The Lonza RNA marker (Lonza Bioscience, USA, #50,577) was used.

Genomic DNA from each alcohol-preserved mosquito was extracted using DNAzol® reagent (Invitrogen, Carlsbad, California, USA). After PCR reactions, the amplified products were analyzed on 3% agarose gel with a low molecular weight DNA ladder (New England Biolab, Ipswich, Massachusetts, USA) used to estimate the band size. The electrophoresis was run for 50 min at 100 V in TBE buffer. The gel was then submerged in 0.5 μg/ml ethidium bromide (EtBr) (Invitrogen) solution for 15 min, de-stained for 5 min in distilled water, and visualized in a UV transilluminator.
Since EtBr is known to be a strong mutagen and is treated as hazardous waste, as alternatives we tried using Ultrapower™ (BioTeke, Beijing, China) and RedSafe™ (iNtRON Biotechnology, Gyeonggi-do, Korea) dyes, which are advertised as non-toxic and have no hazard waste. For Ultrapower™ staining, the dye solution (10,000×) was diluted 100-fold in 6× loading dye (New England Biolab), then 1 μl of diluted dye was mixed with 5 μl of PCR product. 1 μl of diluted dye was also added to 5 μl of the DNA ladder before loading on the gel. For RedSafe™ staining, 5 μl of this dye (20,000×) was mixed in with 100 ml molten agarose gel prior to gel pouring. Visualization was done immediately after gel electrophoresis.

To confirm the integrity of Alveofact-coated polyplexes after sonication, a gel retardation assay was performed. A 1% Agarose gel was prepared and stained with ROTI®GelStain Red. Polyplexes were prepared with 300 pmol siRNA at three different Alveofact:PEI ratios (0, 2.5:1 and 5:1). As positive control, polyplexes were treated with 1 USP unit of heparin. 3 μL of low range ssRNA ladder (New England BioLabs, Ipswich, Massachusetts, USA) and 3 μL of siRNA were respectively diluted in 27 μL of RNA free water. 30 μL of each sample were mixed with 5 μL of loading dye (New England BioLabs, Ipswich, Massachusetts, USA), loaded into the slots of a gel, and electrophoresis was run at constant voltage of 200 V for 15 min in Tris-borate EDTA buffer. The gel was visualized using a ChemiDoc MP imaging system (Bio Rad, Hercules, California, USA).

Circularized cDNA was amplified and barcoded (Table S4) by adding 15 μl of PCR master mix (8 μl HF master mix (NEB), 0.8 μl 10 μM reverse barcoding primer, 0.8 μl 10 μM forward barcoding primer, 5.4 μl water) per 1 μl template (1 cycle: 30 s 98°C;; 3-to-7 cycles: 10 s 98°C; 10 s 60°C; 5 s 72°C;; 1 cycle: Hold 4°C). Tubes were taken at the end of 3-4-5-7 cycles. PCR products were mixed with 3μl loading dye (NEB) and run on 8% TBE gel (Invitrogen) (90 V, 95 min) in 1xTBE buffer. The gel was stained with SybrGold (Invitrogen) (5 min, RT) and DNA corresponding to 120-170 nt was excised. For physical disruption, the gel slices were spun through 0.5 mL tubes with holes at the bottoms nested in 1.5 mL tubes. Then 0.67 mL DNA soaking buffer (0.3 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) was added to the gel slurry and tubes were incubated overnight on a rotating wheel.

Total RNA concentration was determined via nanodrop and 0.5–1 µg was mixed with loading dye (New England Biolabs) and resolved via electrophoresis on a 1% agarose 1× TAE (40 mM Tris, 20 mM Acetate and 1 mM EDTA) with SYBR™ Safe DNA Stain (Invitrogen, 1:1000 dilution) gel at 150 V for 20 min then visualised on a Biorad Chemidoc Imaging system (Blue imaging tray). One kb HyperLadder (Bioline) was loaded on each gel as a reference.

In vitro cleavage assays were performed with purified recombinant ThermoCas9. ThermoCas9 protein, the in vitro transcribed sgRNA and the DNA substrates (generated using PCR amplification using primers described in Supplementary Table 2) were incubated separately (unless otherwise indicated) at the stated temperature for 10 min, followed by combining the components together and incubating them at the various assay temperatures in a cleavage buffer (100 mM sodium phosphate buffer (pH = 7), 500 mM NaCl, 25 mM MgCl₂, 25 (V/V%) glycerol, 5 mM dithiothreitol (DTT)) for 1 h. Each cleavage reaction contained 160 nM of ThermoCas9 protein, 4 nM of substrate DNA, and 150 nM of synthetized sgRNA. Reactions were stopped by adding 6X loading dye (NEB) and run on 1.5% agarose gels. Gels were stained with SYBR safe DNA stain (Life Technologies) and imaged with a Gel DocTM EZ gel imaging system (Bio-rad).