GoKawiilDNA and RNA preparation
All DNA oligonucleotides were ordered from IDT (Supplementary Table 1). DNA oligos were resuspended to 200 μM and subjected to 15% denaturing PAGE, extracted from the gel through ultraviolet shadowing, crushed and soaked, ethanol precipitated, quantified by spectrophotometry (Biodrop) and stored at −20 °C. Some DNA oligos were labelled (Supplementary Table 1) using NHS-ester labelling chemistry, as described previously59, and purified by high-performance liquid chromatography.
In vitro transcription of sgRNA
DNA templates required for sgRNA were annealed together under equimolar conditions by heating to 95 °C for 2 min before cooling by −2 °C min−1 to 4 °C in an annealing buffer containing 50 mM Tris-HCl pH 8 and 100 mM NaCl. The annealed dsDNA template was subjected to in vitro transcription containing 500 mM template, 5 mM of each dNTP, 25 mM MgCl 2 , 0.2 U of Escherichia coli inorganic pyrophosphatase, 80 U of RNase inhibitor, 7.5 mM DTT, 1× RNA polymerase buffer and 750 U of RNA polymerase. This reaction was mixed well and incubated at 37 °C overnight. The reaction was subjected to 15% denaturing PAGE and then purified by ethanol precipitation.
Protein expression, purification and fluorescence labelling
WT and dCas9 were gifted by E. Gordon, and they were expressed and purified as described previously16. Dual-labelled Cas9/dCas9 was labelled with a 1:20:40 ratio of Cas9/dCas9:Cy3 maleimide:Cy5 maleimide in Cas9 storage buffer (20 mM HEPES pH 7.5, 500 mM KCl, 1 mM TCEP). The reaction was left for 2 h at room temperature and then overnight at 4 °C in the dark before loading onto the Superdex 200 Increase 10/300 GL size-exclusion column. The labelled fractions were pooled, quantified using the Biodrop spectrophotometer, aliquoted, flash-frozen in liquid nitrogen and stored at −80 °C.
DNA minicircle assembly
Minicircles were assembled using an adapted protocol described previously60. The design of the minicircles included several AT-tracts to promote bendability, as described60,61,62,63. Three main steps were used: first, the ssDNA (126 nucleotides) and splint DNA (29 nucleotides) were heat-annealed together at 95 °C for 3 min and subsequently cooled down on ice for 1 min. The reaction was incubated with T4 DNA ligase (20 U) at 37 °C for 30 min to form a ssDNA minicircle. Secondly, the ssDNA minicircle was subjected to T4 DNA polymerase fill-in, the ssDNA minicircle was diluted into a reaction containing T4 DNA polymerase (120 U) and T4 DNA ligase (16,000 U) in the presence of 100 μM dNTP mix and 10 mM ATP, and the reaction was incubated at 12 °C for 1 h. Finally, the reaction was treated with Exo V (20 U) and T5 Exo (20 U) in the presence of 1 mM ATP at 37 °C for 45 min; this was done to remove all incompletely assembled minicircles. To clean the reaction further, it was treated with proteinase K to remove any remaining protein in the reaction before passing the reaction through a Monarch PCR & DNA Clean-up Kit (NEB).
Negative supercoiling of DNA minicircles
Minicircles were subjected to negative supercoiling using E. coli gyrase (Inspiralis) in the presence of EtBr. A reaction of 10 nM minicircle DNA (mcDNA), 1× gyrase buffer, 0.1 mg ml−1 EtBr and 20 U μl−1 of E. coli gyrase was incubated at 37 °C for ≥1 h. The reaction was then cleaned either by phenol–chloroform extraction followed with ethanol precipitation (AFM) or using the Monarch PCR & DNA Clean-up Kit (Bulk/Cryo-EM).
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