Ambion megascript t7

Synthesis of the sgRNA specific to each mutation was carried out using the Ambion MEGAscript T7 or SP6 kits (Ambion). Cas9 mRNA was synthesized by linearizing pCS2-nCas9n (Addgene #47929) with the restriction enzyme NotI and subsequently using the linearized plasmid as template for the SP6 mMessage Machine kit (Ambion) to produce capped mRNA. Purification of the in vitro synthesized mRNA was achieved with the RNeasy Mini Kit (Qiagen).

A target site within the abcc9 open-reading frame was chosen using CHOPCHOP (http://chopchop.cbu.uib.no/). sgRNA template was purchased at Integrated DNA Technologies (IDT) as standard desalted same-day oligos and synthesis was carried out using the Ambion MEGAscript T7 or SP6 kits (Ambion). Purification of the in vitro synthesized mRNA was achieved with the RNeasy Mini Kit (Qiagen)^{64 (link)}. gRNA and Cas9-encoding mRNA were co-injected into one-cell stage zebrafish embryos. Each embryo was injected with 2 nl of solution containing ~12.5 ng μl⁻¹ of sgRNA and ~300 ng μl⁻¹ of Cas9 mRNA. Injected embryos were grown to adulthood to generate “founder” fish (F0), and screened for germline transmission of CRISPR-induced indel mutations using an adapted method for detecting simple sequence-length polymorphisms (SSLPs). Each putative founder adult fish was crossed with a wild-type adult fish (F1). Sequencing analysis confirmed successful introduction of 13n deletion and segregation in a Mendelian fashion. Homozygous fish (F2) were generated by inbreeding heterozygous mutant carriers.