Maxiscript t7 kit

Cas9 mRNA was obtained by in vitro transcription with the linearized plasmid pXT7-Cas9 according to the procedure previously described. The sgRNAs were transcribed from the DNA templates that amplified by PCR with a pT7 plasmid as the template, a specific forward primer and a universal reverse primer. The gRNA sequence is listed in the following: 5’-TAATACGACTCACTATAGGTGATTAAAACCGGAGAGGGTTTTAGAGCTAGAAATAGC-3’. Cas9 mRNAs were synthesized in vitro using the linearized constructs as templates with SP6/T7 mMESSAGE mMACHINE Kit (Thermo Fisher Scientific, USA), purified with RNeasy Mini Kit (Qiagen, Germany), and dissolved in RNase free Ultrapure water (Thermo Fisher Scientific, USA). The sgRNAs were synthesized by the MAXIscript T7 Kit (Thermo Fisher Scientific, USA), and were purified with RNeasy Mini Kit (Qiagen, Germany), and dissolved in RNase free Ultrapure water (Thermo Fisher Scientific, USA). Zebrafish lines were naturally mated to obtain embryos for microinjection. One to two-cell stage zebrafish embryos was injected with 2–3 nl of a solution containing 250 ng/μl Cas9 mRNA and 15 ng/μl sgRNA. At 24 h post fertilization (hpf), zebrafish embryos were randomly sampled for genomic DNA extraction according to the previous methods to determine the indel mutations by sequencing.

Guide RNA target sequences were designed with computational tools [44 (link), 45 (link)] (http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design or http://genome-engineering.org) and top predictions per each candidate gene were selected for functional testing (S10 Table). Single guide RNAs (sgRNA) for C6, Ces3b, Itgb6, and Sntg1 were in vitro synthesized (MAXIscript T7 Kit, Thermo Fisher) from double stranded DNA templates by GeneArt gene synthesis service (Thermo Fisher) while the 4 sgRNAs for Arl6ip5 were in vitro synthesized using the Guide-it sgRNA In Vitro Transcription Kit (Clontech). The sgRNAs were purified prior to activity testing (MEGAclear Transcription Clean-Up Kit, Thermo Fisher). Both the Wash and Elution Solutions of the MEGAclear Kit were pre-filtered with 0.02 μm size exclusion membrane filters (Anotop syringe filters, Whatman) to remove particulates from zygote microinjection solutions, thus preventing microinjection needle blockages.

The targeted genomic sequences and oligonucleotides used in this study are listed in Supplementary Table S3. To construct the pDR274-pax2a-gRNA1–3, sense and anti-sense oligonucleotides were annealed and inserted into the BsaI-cleaved pDR274 vector19 (link). The Cas9 plasmid pCS2-hSpCas9 was kindly provided by Dr. Kinoshita (Kyoto University)23 (link). The gRNA was transcribed from the linearized pDR274-derived vector using MAXIscript T7 Kit (Thermo Fisher Scientific). The Cas9 mRNA was transcribed from the linearized pCS2-hSpCas9 using mMESSAGE mMACHINE SP6 Kits (Thermo Fisher Scientific).

Total RNAs were extracted from fly heads using Trizol reagent (ThermoFisher; #15596026), following the manufacturer’s protocol. RNA quality was checked by gel, and 0.6–3 μg of total RNA was loaded in 15% TBE-urea gel (ThermoFisher; #EC68852BOX). RNAs were then transferred onto nylon membrane (GE HealthCare; #RPN303B) and cross-linked by ultraviolet (UV). The membrane was then prehybridized by UltraHyb Oligo Hybridization Buffer (ThermoFisher; #AM8663) and then hybridized with P³²-labeled probes overnight at 50 °C. To make the probes, DNA oligos were annealed to obtain the template for RNA probes, which were synthesized by in vitro transcription using MAXIscript T7 kit (ThermoFisher; #AM1312), supplemented with P³²-α-UTP. Signals were detected by GE Amersham Typhoon 9410 Imager and analyzed by ImageJ for quantification. Probes used are listed in Supplementary Table 4. The same blots were used to probe 2 different snRNAs: either U1 and U11; U2 and U5; U4 and U7 or U4atac and U12. The uncropped scans of the blots are shown in Supplementary Fig. 8.