Tracrrna

Genome editing was performed using CRISPR/Cas9 as described (Paix et al., 2015 (link)). Briefly, purified Cas9 (NEB), tracrRNA, dpy-10 and alg-1 crRNA, and repair templates were incubated at 37°C for ten minutes for in vitro assembly, then loaded for injection. Worms were screened for rollers, bred to separate dpy-10 from alg-1 edits, and sequenced. tracrRNA was purchased from Dharmacon (Lafayette, CO). The sequences of the tracrRNA and repair template sequences are provided in the Key Resources Table.

174CRISPR RNA and tracrRNA (IDT cat# 1072532) were purchased from IDT and annealed to form crRNA174:tracrRNA RNA duplexes according Alt-R CRISPR-Cas9: In vitro cleavage of target DNA with ribonucleoprotein complex protocol (IDT). All RNA oligonucleotides used in this study are listed in Supplementary Data File 2.Target DNA substrates were generated by PCR of purified φNM4γ4 genomic DNA with Phusion DNA Polymerase (Thermo) using oPN144 and oPN562 and then purified by gel extraction. Cleavage assays were preformed similarly as described previously (Jinek et al., 2012 (link)). Briefly, crRNA:tracrRNA and Cas9 (NEB #MO386) were allowed form RNP complexes at room temperature for 10 minutes and then diluted to a final concentration of 6.25, 12.5, 25, 50 and 100 nM following to the addition of target DNA. All reactions were incubated at 37°C for 5 minutes before the digestion with proteinase K (NEB #P8107) to stop the reactions and liberate target DNA and then stored at −80°C until ready for further analyzed. Samples were visualized on 2% agarose gel with SYBR Gold Nucleic Acid Gel Stain (Invitrogen #S11494) and the abundance of cleavage products quantified by automated electrophoresis and imaging using a Tapestation 4200 (Agilent).