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Brain transcriptome profile after CRISPR-induced ghrelin mutations in zebrafish


Ghrelin (GRL) is a gut-brain hormone with a role in a wide variety of physiological functions in mammals and fish, which points out the ghrelinergic system as a key element for the appropriate biological functioning of the organism. However, many aspects of the multifunctional nature of GRL remain to be better explored, especially in fish. In this study, we used the CRISPR/Cas9 genome editing technique to generate F0 zebrafish in which the expression of grl is compromised. Then, we employed high-throughput mRNA sequencing (RNA-seq) to explore changes in the brain transcriptome landscape associated with the silencing of grl. The CRISPR/Cas9 technique successfully edited the genome of F0 zebrafish resulting in individuals with considerably lower levels of GRL mRNAs and protein and ghrelin O-acyl transferase (goat) mRNAs in the brain, intestine, and liver compared to wild-type (WT) zebrafish. Analysis of brain transcriptome revealed a total of 1360 differentially expressed genes (DEGs) between the grl knockdown (KD) and WT zebrafish, with 664 up- and 696 downregulated DEGs in the KD group. Functional enrichment analysis revealed that DEGs are highly enriched for terms related to morphogenesis, metabolism (especially of lipids), entrainment of circadian clocks, oxygen transport, apoptosis, and response to stimulus. The present study offers valuable information on the central genes and pathways implicated in functions of GRL, and points out the possible involvement of this peptide in some novel functions in fish, such as apoptosis and oxygen transport.

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This study was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2016-74857-C3-2-R and AGL2016-74857-C3-3-R) to MJD and JMCR. AMB was a predoctoral fellow funded by the Spanish Ministry of Education and Science when this study was carried out.

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Correspondence to José Miguel Cerdá-Reverter or María Jesús Delgado.

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José Miguel Cerdá-Reverter and María Jesús Delgado are equal co-senior contributing authors.

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Fig. A1

PAGE electrophoresis showing the genotyping of grl sgRNA/Cas9-injected and uninjected zebrafish embryos based on the characterization of the duplex formation during DNA denaturation and annealing. Only homoduplexes are observed in the uninjected (control, CTRL) embryos, while homoduplexes and heteroduplexes are detected in the grl sgRNA/Cas9-injected (INJ). MM, molecular marker (PNG 51 kb)

High Resolution Image (TIF 406 kb)

Table A1

Differentially expressed genes between grl KD and WT fish after RNA-seq analysis (PDF 1412 kb)

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Blanco, A.M., Cortés, R., Bertucci, J.I. et al. Brain transcriptome profile after CRISPR-induced ghrelin mutations in zebrafish. Fish Physiol Biochem 46, 1–21 (2020).

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  • Ghrelinergic system
  • CRISPR/Cas9
  • Knockdown
  • RNA-seq
  • Transcriptomic
  • Fish