To assemble the SPARSA-gRNA-tDNA complex, the purified SPARSA sample was incubated with 42 nt 5’ phosphorylated gRNA56 (link) at a molar ratio of 1:1.2. After incubation at 4 °C for 30 min, complementary tDNA56 (link) was added at a molar ratio of 1:1.2 and incubated at 4 °C for 30 min. The resulting sample was then subjected to a Superdex 200 Increase column (Cytiva Life Sciences). The fractions were used to perform cryo-EM experiments.
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