Murine rnase inhibitor

For the measurement of the trans cleavage activity of LwaCas13a at the single-molecule level, imaging was immediately started after the hexadecane loading onto the microchamber device. Fluorescence images on a single stage point were recorded at time intervals of 3 s. The number of cleaved FQ reporters was calculated based on the mean fluorescence intensity in each chamber, using a calibration curve of mean fluorescence intensity to FAM concentration (Supplementary Fig. 4), and the chamber volume was measured by a laser microscope (Keyence). To obtain the calibration curve, fluorescein-conjugated ssRNA without any quenchers (56-FAM/rUrUrUrUrU, Integrated DNA Technology) in buffer D, containing 500 μM Triton X-100 and 20 μM Alexa Fluor 647, were loaded into the microchamber device, followed by sealing with hexadecane, and the imaging was conducted using the same setups as in the SATORI assay.
Bulk trans cleavage assays were performed, using LwaCas13a (45 nM), crRNA (22.5 nM), FQ reporter (125 nM), murine RNase inhibitor (0.5 μL, New England Biolabs), and various amounts of tgRNA in assay buffer (20 mM HEPES (pH 6.8), 60 mM NaCl, and 6 mM MgCl₂). Reactions were incubated at 37 °C for 10 min, and fluorescence was detected at every 1 min at excitation (470 nm) and emission (520 nm) wavelengths on a fluorescence microplate reader (SpectraMax ID3, Molecular Devices).

All primer sequences used in this study are available in Supplementary Data Sheet 1. Reverse transcription was performed on 500 ng of total pokeweed RNA in a 20 μL reaction volume containing 5 mM DTT, 1 μM reverse primer, 1X First Strand Buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂), 0.5 mM dNTPs, 20 units Murine RNase Inhibitor (NEB) and 25 units Superscript III reverse transcriptase (Thermo Fisher). The reaction was incubated at 42°C for 1 h and heat inactivated at 70°C for 20 min.
Following cDNA synthesis, a PCR reaction was conducted, containing 1X Q5 buffer (NEB), 0.5 μM forward primer, 0.5 μM reverse primer, 200 mM dNTPs, 2 μL cDNA and 1 unit Q5 DNA polymerase (NEB) in a total volume of 50 μL. The PCR program included an initial denaturation of 95°C for 30 s, 30 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 120 s and finished with an extension at 72°C for 180 s. PCR products were separated on low-melt agarose and the correct size band excised and purified with EZ-10 Spin columns (Biobasic). The purified product was digested with BamHI and SalI, ligated into pBS-KSII and sequenced.

For estimation of the mRNA level of eol-1, nduo-5, and ctb-1, around 200 L4 larvae were handpicked from mixed population and frozen by liquid nitrogen. The total RNA was isolated by TRIzol extraction (Thermo Fisher, Waltham, Massachusetts, United States of America, #15596026). For analyses of cytosolic mRNA level of nduo-5 and ctb-1, approximately 2,000 worms were synchronized by bleach preparation of eggs, hatching progeny eggs to L1 larvae, grown until L4 stage, and frozen by liquid nitrogen. Worm lysates were generated by TissueLyser with steel beads (Qiagen, Venlo, the Netherlands, #69989), and resuspended in 500 μl of the lysis buffer containing 0.8-M sucrose, 10-mM Tris-HCl, 1-mM EDTA, 1× protease inhibitor cocktail (Roche, Basel, Switzerland, 11873580001), and 1-U/μl murine RNase inhibitor (New England BioLabs, Ipswich, Massachusetts, USA, #M0314L). The lysate was centrifuged at 2,500 × g for 10 min at 4°C to remove the pellet debris. And the supernatant was centrifuged at 20,000 × g for 10 min at 4°C. Collect the pellet for western blot, and the supernatant was centrifuged at 20,000 × g for 10 min at 4°C. Take 100 μl of the supernatant for western blot and 300 μl for RNA isolation with TRIzol.

Fluorescent proteins were synthesized from single DNA molecules using PURExpress in vitro protein synthesis kit (NEB) in the femtoliter droplet array (FemDA). 6 μl solution A, 4.5 μl solution B, 0.3 μl murine RNase inhibitor (NEB), 2.7 μl nuclease-free water, and 1.5 μl pre-diluted (i.e., 100-fold in nuclease-free water) DNA sample were mixed (total 15 μl), resulting in a total dilution factor of 10³ for the DNA digest. For a multiplexed assay, each DNA sample was 1 μl, and the water volume was accordingly adjusted to maintain the total volume unchanged. This mixture solution was introduced into the microchannel. A sequential injection of AE-3000 (spiked with 0.1 wt% Surflon S-386, AGC) and Fomblin Y25/6 oil (Solvay) isolated and sealed microchambers, forming the femtoliter droplets. The CFPS reaction was carried out at room temperature.

Prior to cDNA synthesis, RT primers (Table S1) were mixed equimolarly, and the concentration of each primer in the mix was 2 µM. 1 µL of RT primer mix was annealed to 2 µg of RNA in 5.9 µL water at 65 °C and immediately placed on ice. Next, 100 units of TGIRT-III (InGex) reverse transcriptase in TGIRT buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂), 0.5 µL of 100 mM freshly prepared dithiothreitol, and 4 units of Murine RNase Inhibitor (NEB) were added to primer annealed RNA, and pre-incubation was conducted at 25 °C for 30 min. Reverse transcription was initiated by addition of 1 µL of 10 mM dNTPs and performed for 2 h at 57 °C. The final volume of the reaction was 10 µL. RNA was removed by adding 5 units of RNase H (NEB) and incubating for 20 min at 37 °C. RNase H was inactivated by 20min incubation at 65 °C. cDNA was purified using 2.5X strength Ampure XP beads (Beckman Coulter) and resuspended in 100 µL of RNase-free water (EURx).

Lysis buffer was made fresh each time and comprised 1× gradient buffer (150 mM NaCl, 15 mM MgCl₂, 15 mM Tris-HCl pH 7.5, 100 µg/mL cycloheximide (the gradient buffer can be stored at −20 °C), 1% Triton X-100, 0.05% Tween-20, 0.5 mM DTT, 5 mM NaF, 1× cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland), 1× Protease Inhibitor Cocktail for plant cell and tissue extracts (Merck), 2% n-Dodecyl-beta-Maltoside Detergent (Thermo Scientific, Waltham, MA, USA). For lysis tests, 200 µL lysis buffer (with either 1000 U/mL Ribolock (Thermo Scientific), 1000 U/mL Superase.In (Invitrogen, Waltham, MA, USA), 1 mg/mL Heparin (Merck, Darmstadt, Germany), 1000 U/mL, Murine RNase Inhibitor (New England Biolabs, Ipswich, MA, USA), or 10 mM Ribonucleoside Vanadyl Complex (New England Biolabs) was aliquoted and chilled on ice. A total of 20 µL of ground tissue sample was then tipped straight from dry ice into the lysis buffer. Tubes were immediately inverted to disperse the tissue fragments. Lysates were incubated on ice for 5 min with occasional inversion, then centrifuged at 13,000 rpm, 4 °C, for 5 min. A total of 150 µL of supernatant was retained and 1 mL TRIzol added (Invitrogen).