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Cas9 and RNA preparation

CR C. D. Richardson
GR G. J. Ray
NB N. L. Bray
JC J. E. Corn
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Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal peptides and an HA tag at the C terminus was expressed in Rosetta2 DE3 (UC Berkeley Macrolab) cells. Cell pellets were sonicated, clarified, Ni2+-affinity purified (HisTraps, GE life sciences), TEV cleaved, cation-exhanged (HiTrap SP HP, GE life sciences), size excluded (Sephacryl S-200, GE life sciences) and eluted at 40 μM in 20 mM HEPES KOH pH 7.5, 5% glycerol, 150 mM KCl, 1 mM dithiothreitol (DTT)20. sgRNAs were generated by HiScribe (NEB E2050S) T7 in vitro transcription using PCR-generated DNA as a template (dx.doi.org/10.17504/protocols.io.dm749m). Complete sequences for all sgRNA templates can be found in Supplementary Data 2.

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