RT Dissertation/Thesis
T1 CRISPR-RfxCas13d optimizations and functional screenings identify Bckdk as a novel kinase regulating the maternal-to-zygotic transition in teleosts
A1 Hernandez Huertas, Luis
K1 Genética del desarrollo
K1 Biología molecular
AB The maternal-to-zygotic transition (MZT) is a universal and reprograming process that occurs during early animal development encompassing the activation of a silent zygotic genome and the clearance of the maternally provided RNAs. While some maternal transcriptional factors and chromatin modifiers regulating zygotic genome activation (ZGA) and MZT have been identified, there are thousands of deposited maternal RNAs which no function has yet been assigned. In particular, post-translational modifiers have been poorly characterized in the context of MZT. Consequently, to comprehensively understand this fundamental regulatory mode during early development, a systematic analysis of the maternal contribution (protein, but especially RNA) is required. In this work we have optimized CRISPR-RfxCas13d system in zebrafish embryos through transient approaches such as ribonucleoprotein (RNP) or mRNA-RNA complexes and used it to efficiently perform a knockdown (KD) screening targeting maternal mRNAs encoding protein kinases and phosphatases. Among them, the depletion of bckdk mRNA, a mitochondrial and cytosolic kinase, experienced the most severe epiboly defects, a phenotype compatible with an MZT alteration. RNA-Seq, SLAM-Seq, ATAC-Seq and biochemical approaches upon cytosolic Bckdk depletion revealed i) a general deregulation of the ZGA, ii) an inefficient miR-430 maternal RNA clearance, and iii) a slight chromatin accessibility alteration but significant H3K27ac depletion. Furthermore, a phospho-proteomic analysis uncovered that Phf10/Baf45a, a chromatin remodelling factor, is less phosphorylated upon bckdk mRNA depletion. Further, phf10 mRNA KD also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after the depletion of bckdk mRNA. Finally, we validated the role of Bckdk during MZT in medaka, another teleost model. Additionally, we have implemented various and compatible strategies to enhance transient CRISPR-RfxCas13d RNA targeting in vivo. Firstly, to streamline the prediction and selection of highly efficient gRNAs, we tested in vivo three different computational models developed recently to predict CRISPR-RfxCas13d activity in vitro, employing data from numerous KD events in zebrafish embryos. Secondly, we demonstrated that chemically modified gRNAs, in combination with RfxCas13d mRNA, enhance and sustain mRNA KD in zebrafish embryos, especially for genes with late expression during early development. Finally, we optimized RfxCas13d nuclear targeting in vivo by incorporating refined nuclear localization signals from other CRISPR-Cas system. Altogether, our results demonstrate the potential of CRISPR-RfxCas13d system to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.
YR 2025
FD 2025
LK https://hdl.handle.net/10433/24554
UL https://hdl.handle.net/10433/24554
LA en
NO Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biología Celular, Molecular e Ingeniería GenéticaClave Programa: DBICódigo Línea: 108
NO Universidad Pablo de Olavide. Escuela de Doctorado
DS RIO
RD May 4, 2026