Dna rna chimeric oligonucleotides

DNA oligonucleotides and DNA–RNA chimeric oligonucleotides were synthesized by Integrated DNA Technologies (IDT). Chimeric crRNAs were prepared for RNP assembly by heating to 95°C then placing on ice to prevent formation of stable secondary structures. Cas9 tracrRNAs were prepared by T7 RNA polymerase in vitro transcription with DNA templates synthesized by IDT. Single-stranded DNA templates were annealed to T7 promoter oligo to generate double-stranded promoter regions, which support in vitro transcription by T7 RNA polymerase. In vitro transcriptions were performed by standard protocols for 2 h. Briefly, reactions contained purified T7 RNA polymerase (4 μM), 30 mM Tris (at pH 7.9), 12.5 mM NaCl, 40 mM MgCl2, 2% PEG 8000, 0.05% Triton X-100, 2 mM spermidine and 2.5 μM T7-DNA template. Afterward, the DNA template was degraded by DNase I treatment. Reactions were phenol–chloroform extracted and gel-purified from denaturing polyacrylamide gels. Purified RNA was refolded by heating to 95°C for 5 min in a heating block followed by slow cooling the block to RT (∼40 min). RNA was quantified by measuring absorbance at 260 nm and calculated extinction coefficients using nearest neighbor approximations and Beer’s Law.

DNA and DNA–RNA chimeric oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Table S2). Fluorophore and quencher labelled sequences were purified by HPLC while other sequences were purified through standard desalting method. All oligonucleotides were reconstituted in DI H₂O and stored in −20 °C freezer as recommended by manufacturer and used without further purification. UV-Vis spectra were taken to calibrate the concentration of all oligonucleotide-containing solutions prior to usage.