CRISPR Guide RNA Preparation Protocol

Custom crRNA (IDT, Alt-R^TM crRNA) and generic tracrRNA (IDT, 1072532) were resuspended to 100 μM in sterile and nuclease-free T₁₀E_0.1 buffer (10 mM Tris-HCl, 0.1 mM EDTA, embryo-tested water (Sigma, W1503)) prepared as described [16 (link)]. 50 μM crRNA:tracrRNA complexes (pgRNA) were generated by subjecting equimolar ratios to 95°C for 5 min followed by reduction of 5°C/min. sgRNA was generated by T7 RNA polymerase mediated in vitro transcription (NEB, E2040S) from the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene #48138) from Feng Zhang and column purified (Qiagen, 217004). Guide RNAs were stored at -80°C. crRNA sequences are listed in S1 Table.