sgRNA Synthesis
Crisprscan36 was used to identify a target site in exon 3 of exosc9 in zebrafish. Single guide RNA (sgRNA) was produced as described elsewhere.37 An oligonucleotide with a T7 promoter, exosc9 target sequence, and a complementary sequence (3′-taatacgactcactataGGGGGGCGTGAATCTTTTGGgttttagagctagaa-5′) was annealed to a bottom strand “ultramer” oligo (3′-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGG ACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-5′) in a thermocycler, and extension of the oligonucleotides was catalyzed by DNA polymerase (MyTaq) to form the template oligonucleotide for sgRNA synthesis.38 The sgRNA template oligonucleotide was purified with a Qiagen PCR Purification Kit. sgRNA was synthesized from the sgRNA oligonucleotide template with the MEGAshortscript T7 Kit (Ambion), purified with the mirVana RNA Isolation Kit (ThermoFisher Scientific), and stored at −80°C until required for injection.