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Fig. 5
Crispant analysis of transcription factor binding sites upstream of myl7. (A) The zebrafish myl7 locus encoding cardiac myosin light chain 7, with an enlarged depiction of the upstream regulatory region and putative GATA and MZF transcription factor binding sites. (B) Panel plot depicting Cas9-mediated mutagenesis of the GATA site region in individual representative crispants for the endogenous myl7 locus and the single-copy myl7:EGFP transgene. (C-J) Anterior lateral views at 36hpf of heterozygous transgenics for myl7:EGFP targeted with sgRNAs recognizing the GATA or MZF binding site or the interspersed region between the two sites (INTER) in myl7 with solubilized RNPs. (C,D) Control sibling with EGFP expression in the heart tube. (E,F) Reduction or loss of EGFP upon targeting of the GATA site with sgRNA[GATA], (G,H) the MZF site using sgRNA[MZF] or (I,J) the interspersed sequence with sgRNA[INTER]. Arrowheads indicate cardiac edema (E,G); asterisks indicate impaired EGFP signal (F,H). (K-N) mRNA in situ hybridization (ISH) for endogenous myl7 expression at 36hpf in examples of control siblings (K), GATA site crispants (L,M) and MZF site crispants (N). (K) A, atrium; V, ventricle. Arrowheads indicate decreased myl7 expression (L,N); asterisk indicates complete loss of ISH signal (M). (O) Phenotype penetrance and expressivity of myl7 ISH signal in transcription factor site crispants. (P-S) Germline transmission of GATA site mutations. (P,Q) Control myl7:EGFP sibling. (R,S) GATA site germline mutant from crispant incross, featuring cardiac edema (arrowhead, R) and impaired EGFP expression (asterisk, S). (T) Allele sequences of endogenous and hemizygous transgene locus in an F1 embryo, featuring lesions in the predicted GATA site.