Genetically modified zebrafish. (A) Transient mosaic expression of FLAG-tagged MyBP was accomplished by transposase-mediated random integration of exogenous MyBP cDNA. We used the modular “tol2kit” approach to assemble the transgene constructs as described in Materials and methods. Briefly, pTol2-Hsp70I:mybphb-3XFLAG and pTol2-hsp70I:mybpc2b-3XFLAG plasmids were generated by cloning chemically synthesized transgene cDNA, together with hsp70I promoter and SV40 late poly-A sequences, into a backbone vector containing tol2 ITR sequence and a cardiac-specific eGFP reporter cassette. (B) The mybphb null allele (Figs. 3, 4, and 5) was generated by creating double-strand breaks in exons 1 and 4 using spCas9 protein precomplexed to guide RNAs to those exons (see Table S5), followed by cell-mediated repair and excision of the intervening ∼12 kb of genomic DNA. Genotyping was accomplished by PCR using primers a, b, c, as shown (see Table S5). Precise characterization of the mutant allele was accomplished by PCR followed by Sanger sequencing. (C) The SA10810 mutation in mybpc2b (Fig. 6) consists of a single base substitution in exon 7 creating a premature STOP codon. Genotyping was accomplished by PCR and bidirectional Sanger sequencing of the mutation site (see Table S5). Source data are available for this figure: SourceData FS1.