Pt3ts ncas9n plasmid

Injection of p53 morpholino (MO) or esco2 Cas9/guide RNA was performed on one-cell-stage zebrafish embryos at a concentration using 0.5 nl of 0.85 mM. Injected embryos were incubated at 28°C until the indicated stage and analyzed via brightfield microscopy. The sequence of p53 MO used to target exon 2 splice donor site of the p53 gene was 5′-CCCTTGCGAACTTACATCAAATTCT-3′. Cas9 mRNA was transcribed from the linearized pT3TS-nCas9n plasmid (Addgene) using the mMessage mMachine T3 kit (Life Technologies). Each RNA was purified using the RNeasy Kit (Qiagen). The CRISPR guide RNA was synthesized using the MegaShortScript T7 Kit (Life Technologies) and purified using the MegaClear Kit (Life Technologies). RNA concentration was quantified using the Nanodrop spectrophotometer. For CRISPR/Cas9 injections, 150 ng/µl of Cas9 mRNA and 30 ng/µl of RNA were used (Thomas et al., 2014 (link)).

Two sgRNAs targeting the single zebrafish taf1 gene with no predicted off-target effects were designed using the online software CHOPCHOP^{51 (link)}: one targeting exon 7 (5′-GGG CTA AGA AAA AAT CAG GGT GG-3′) and the other targeting exon 8 (5′-GGT GTC CCG GAG GAT GGC AGC GG-3′). The sgRNAs were prepared as previously described^{52 (link)}, creating a fragment consisting of the T7 promotor, the targeted gene specific sequence, and the guide core sequence. The sgRNAs were synthesized by in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA). Cas9 mRNA was prepared by in vitro transcription with the mMESSAGE mMACHINE T3 Transcription Kit (Life Technologies, Carlsbad, CA) using 500 ng of linearized plasmid that was retrieved from 5 μg of p-T3TS-nCas9n plasmid (plasmid #46757; Addgene, Cambridge, MA) digested with XbaI (New England Biolabs, Ipswich, MA). The products were purified, and their integrity were assessed using a denaturation gel.

Two sgRNAs targeting the single zebrafish nkx3.2 gene with no predicted off-target effects were designed using the online software CHOPCHOP [29 (link)], both targeting the first exon: 5´-GATCAGGAATCCGCGGCCAA-3´ and 5´-GTCGTTGTCCTCGCTCAGCC-3´. The second base of each target was modified to "G" in order to allow T7 transcription without modifications. The sgRNAs were prepared as previously described [30 (link)], creating a fragment consisting of the T7 promotor, the targeted gene-specific sequence, and the guide core sequence. The sgRNAs were synthesised by in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA). Cas9 mRNA was prepared by in vitro transcription with the mMESSAGE mMACHINE T3 Transcription Kit (Life Technologies, Carlsbad, CA) using 500 ng of linearised plasmid that was retrieved from 5 μg of p-T3TS-nCas9n plasmid (plasmid #46757; Addgene, Cambridge, MA) digested with XbaI (New England Biolabs, Ipswich, MA). The gRNAs were purified with mirVana miRNA isolation kit (Life technologies, Carlsbad, CA) and Cas9 mRNA was purified with RNeasy Mini Kit (Qiagen, Maryland; MD), and their integrity was assessed using a denaturation gel.

We generated germline mutant alleles using CRISPR/Cas9 mutagenesis (Hwang et al., 2013 (link)) with modifications as described (Mitchell et al., 2021 (link)). Briefly, we designed sgRNAs(Single-guide RNA) within or just downstream of the MADS or MEF2 domain. The XbaI-digested pT3TS-nCas9n plasmid (Addgene plasmid #46757) was used as a template to transcribe Cas9 mRNA with the T3 mMESSAGE kit (Invitrogen). We transcribed sgRNAs (see table below) from PCR-generated templates using the MEGAscript T7 Kit (Thermo Fisher Scientific). One cell-stage embryos were injected with a mix of 200 ng/µl Cas9 mRNA and 50 ng/µl of each gene-specific sgRNA. Injected embryos were raised, and founders identified by amplifying the genomic region containing the sgRNA site and identifying banding size shifts indicating insertions and/or deletions (see genotyping assay table for primers). All new paralog mutants were originally generated in the low-penetrance strain and were subsequently maintained on the AB background for at least three generations. The following sgRNAs were used: mef2d^co3013: 5’-GGACAAATACCGGAAGAGCG-3’; mef2b^co2012: 5’-CACGAGAGCCGCACTAACAC-3’;mef2aa^co3017: 5’-TCATGGACGACCGTTTCGGC-3’.We generated six independent sgRNAs for mef2ab, and none of them mutagenized this locus. Precise sequences of mutant alleles are indicated below:

TALEN expression vectors were linearized with SmaI and transcribed in vitro using the mMessage mMachine T7 and Poly(A) Tailing Kit (ThermoFisher). The pT3TS-nCas9n plasmid (Addgene), linearized using XbaI and purified [18 (link)], was used in an in vitro transcription reaction (T3 mMessage mMachine, ThermoFisher). The runx3 guide RNA was synthesized using the MegaScript T7 Kit (ThermoFisher). RNA products were cleaned by phenol-chloroform and isopropanol precipitation. The TALEN mixture containing equal amounts of each mRNA (400 pg each) was injected into one-cell stage AB strain zebrafish embryos. The CRISPR/Cas9 injection contained 300pg of Cas9 mRNA and 50ng of runx3 gRNA and was also injected into one-cell stage zebrafish embryos. Injected embryos were raised to adulthood, outcrossed, and gDNA isolated from F1 embryos to identify mutants.