Ntp mix

T7::sgRNA expression cassettes were amplified from their respective pJET1.2 host vectors using primers listed in Additional file 1: Table S3. One microgram of gel-purified linear DNA was used as a template in an in vitro transcription reaction using 100 U of T7 RNA Polymerase (EP0111, Thermo Fisher), 0.1 U inorganic pyrophosphatase (EF0221, Thermo Fisher), 40 U RiboLock RNase Inhibitor (EO0381, Thermo Fisher), and 10 mM NTP mix (R0481, Thermo Fisher) in 1× T7 RNA Polymerase buffer for 16 h at 37 °C. Transcribed sgRNAs were purified by phenol - chloroform extraction.

The plasmids containing the SINV infectious clone were purified by the Plasmid Maxi Kit (Qiagen, Hilden, Germany), linearized by digestion with NotI (New England Biolabs, Ipswich, MA, USA), and purified by phenol-chloroform extraction. RNAs were then synthesized in an in vitro transcription system by incubating at 37°C for 1 h containing the linearized DNA template, NTP mix, dithiothreitol, RNaseOUT ribonuclease inhibitor, and SP6 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Following DNase I treatment, LiCl Precipitation Solution (Invitrogen) was added to purify the synthesized RNAs.

In vitro transcription was performed as previously described^{19 (link)}, with slight modifications. Briefly, PT-modified oligonucleotides (ssDNA) were chemically synthesized (Sangon Biotech, Shanghai, China), and annealed to obtain PT-modified dsDNA; non–PT-modified DNA templates were generated by PCR amplification. Both DNA templates were purified using a GenElute PCR Clean-Up Kit (Sigma, St. Louis, USA) and then quantified by using an Invitrogen Qubit dsDNA High-Sensitivity (HS) Assay Kit (Thermo Fisher Scientific, MA, USA). Afterwards, 100 ng of DNA template was transcribed in a 20-μl reaction mixture that included 2 units of E. coli RNA polymerase, holoenzyme (New England Biolabs, MA, USA), 4 μl of 5× E. coli RNA polymerase reaction buffer, 0.5 mM NTP Mix (Thermo Fisher Scientific, MA, USA), and 20 units of RNase inhibitor (Solarbio, Beijing, China). In vitro transcription was conducted at 37 °C for 12 h, after which the temperature was increased to 85 °C for 10 min to stop the reaction. The RNA product was quantified by using an Invitrogen Qubit RNA HS Assay (Thermo Fisher Scientific, MA, USA).