Efficient Generation of CRISPR sgRNAs in Zebrafish

sgRNA DNA template was generated by fill in PCR (Supplementary Fig. 1c and d). Briefly, a 52 nt oligo (sgRNA primer), containing the T7 promoter (Supplementary Table 1 #1), the 20 nt of the specific sgRNA DNA binding sequence and a constant 15 nt tail for annealing, was used in combination with a 80 nt reverse oligo to add the sgRNA invariable 3′ end (tail primer). A 117 bp PCR product was generated following these parameters: 3 minutes at 95°C, 30 cycles of 30 seconds at 95°C, 30 seconds at 45°C and 30 seconds at 72°C, and a final step at 72°C for seven minutes. PCR products were purified using Qiaquick (Qiagen) columns and approximately 120–150 ng of DNA were used as template for a T7 In vitro transcription (IVT) reaction (AmpliScribe-T7-Flash transcription kit from Epicentre) (Supplementary Fig. 1c). In vitro transcribed sgRNAs were DNAse treated and precipitated with Sodium Acetate/Ethanol. Alternative sgRNAs were generated similarly using shorter (50 or 51 nt) and longer (53 or 54nt) sgRNA primers, with 18–22nt complementary to the target. SgRNAs beginning with GA sequences contained the SP6 promoter (Supplementary Table 1 #2) instead of the T7 promoter. A MAXIscript SP6 transcription Kit (Life technologies) was used for SP6 based IVT reactions.
Zebrafish codon-optimized protein from pT3TS-nCas9n (#2656)^{37 (link)} was used in all experiments except for the pull down, where FLAG-Cas9 was employed. A N-terminal 3xFLAG-tag was cloned in pT3TS-nCas9n in the NcoI site. The resulting pT3TS-FLAG-nCas9n (#2722) plasmid is identical to one previously used for a similar experiment in cell lines^{4 (link), 12 (link)}. For the cas9-nanos 3′-UTR construct, the nanos 3′-UTR and SV40 late polyA signal was PCR amplified from a previous plasmid pCS2+GFP-nanos 3′UTR^{18 (link), 19 (link)} using two oligos (Supplementary Table 1 #3 #4). The following PCR product was then digested in 3′ with NotI and ligated into the pCS2-nCas9n ^{37 (link)} plasmid previously digested with SnaBI and NotI. The final pCS2-nCas9n-nanos 3′UTR (#2662) (addgene #62542) construct was confirmed by sequencing. Cas9 mRNA was in vitro transcribed from DNA linearized by either NotI (pCS2-nCas9n-nanos 3′UTR) or XbaI (pT3TS-nCas9n and pT3TS-FLAG-nCas9n) using the mMachine SP6 or T3 kit (Ambion), respectively. In vitro transcribed mRNAs were DNAse treated and purified using RNeasy Mini Kit (Qiagen).