Determining the function of genes is fundamental for understanding vertebrate development, regulatory
mechanisms and human diseases. Genome editing technologies, such CRISPR-Cas9, have allowed associating
specific phenotypes to permanent gene alteration. However, some key technical and conceptual issues remain
problematic in vertebrates, particularly in aquatic model organisms such zebrafish. For example, the maternally
provided mRNA can rescue the phenotype of homozygous mutants; genotyping steps are tedious; and long non-
coding RNA, lethal or tissue/temporal genes are difficult to study using DNA manipulation as well as complex
genomic loci. Complementary ‘knock-down’ approaches are invaluable tools to circumvent some of these
problems, however, there was no systematic tool to knockdown mRNAs in zebrafish or other teleost fish.
Our long-term goal is to unravel the function of genes related to regulatory mechanisms, development, and
human diseases. Our recent publication demonstrates that injection of the CRISPR-RfxCas13d system into
vertebrate embryos provides a robust and cost-effective technology to systematically disrupt gene function.
However, the injection of this system only provides transient knockdown for ~3 days. Therefore, the central goal
of the proposal is to transfer the CRISPR-RfxCas13d technology from being injected into vertebrate embryos, to
being endogenously expressed through transgenesis. We propose that the development of a repertoire
transgenic zebrafish expressing the RfxCas13d enzyme (ubiquitous and tissues-specific) and guideRNA would
facilitate rapid and vigorous investigation into gene functions. Our preliminary data indicate that transgenic
expression of RfxCas13d in zebrafish is functional and not toxic. The objectives are: 1) Define the optimal system
to knockdown mRNA expression in CRISPR-RfxCas13d transgenic zebrafish. 2) Knockdown mRNA expression
in a tissue-specific manner using specific transgenic lines. This proposal is conceptually innovative as it is based
on the exploration of a novel technique, CRISPR-RfxCas13d, to knockdown mRNA in a tissue-specific manner
in zebrafish. This approach has never been done in vertebrate model systems where RNAi does not work.
The outcomes of this project will be the first transgenic system to dissect and study gene function by knocking
down mRNA expression in zebrafish. Our approach will help the scientific community investigate gene function
in a faster and tissue-temporal specific manner, as well as answering questions that are very challenging to
address by current methodologies, such as the function of non-coding RNAs or phenotypes caused by multiple
genes. Moreover, by following the guidelines we will optimize through this project, researchers may produce a
whole guideRNA collection, targeting all genes, which should be available for anyone to order to address
their individual research questions. Finally, as we have successfully implemented the CRISPR-Cas13d system
by injection in other organisms such as medaka, killifish, and mouse embryos, our work described in this proposal
may serve as the foundation for transferring this efficient knockdown technology into a range of other species.