In vivo detection of messenger ARN molecules using CRISPR technology

SARMIENTO, ANDREA; ABIB, AGUSTINA; AGÜERO, TRISTAN

Congreso; XXXIX ANNUAL SCIENTIFIC MEETING TUCUMAN BIOLOGY ASSOCIATION; 2023

Embryonic development is a complex, strictly regulated and coordinated process that allows each cell and tissue to grow correctly and form a complete organism. This process begins long before fertilization, more precisely during oogenesis, where an accumulation of messenger RNA (mRNA), proteins and mitochondria are precisely located in the oocyte cytoplasm. This enrichment of "maternal content" is critical for the mature oocyte to be fertilized and the resulting zygote to develop correctly. The accumulated maternal transcripts are located in precise and determined sites within the cytoplasm. The correct production, assembly and location of mRNAs are key events required for a normal development. In this work we develop a tool based on CRISPR technology to in vivo visualize mRNA. We used a hybrid Cas13 protein that has its nucleic acid cleavage sites inactivated by mutation fused in-frame to a reporter protein. By abrogating the cleavage sites and preserving the nucleic acid binding sites it is possible to use the Cas13 protein to visualize messenger RNAs in vivo directly in the developing embryo in real time. We designed guide RNAs against the selected genes to use with the mutated Cas13 enzyme and then microinjected them in early staged Xenopus embryos. We detected fluorescence in the primordial germ cells corresponding to the RNA helicase mov10 expression domain and also in the neural plate and neural folds, corresponding to the transcription factor Yy1 expression domain. This innovative technique allows a genuine and reliable analysis of what happens in a dynamic and complex environment such as a living cell and particularly a developing embryo.