Casting CRISPR-Cas13d to fish for microprotein functions in animal development

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Traditional gene annotation used a 100-codon cutoff, potentially missing functional short proteins.
• Recent studies identified numerous short open reading frames encoding biologically active microproteins.
• The functions of most identified microproteins remain largely unknown.

Purpose of the Study:

• To investigate the roles of microproteins in animal development.
• To overcome technical barriers in studying microprotein function, particularly those encoded by maternally provided mRNAs.
• To establish a system for efficient interrogation of microprotein functions during development.

Main Methods:

• Utilized functional genetics, ribosome profiling, and proteomic profiling to identify short open reading frames and microproteins.
• Employed the RNA-targeting CRISPR-Cas13d system in zebrafish.
• Leveraged CRISPR-Cas13d to achieve potent knockdown of targeted mRNAs, including maternally provided ones.

Main Results:

• Demonstrated that the CRISPR-Cas13d system effectively targets and knocks down maternal and zygotic mRNAs in zebrafish.
• Showcased the system's capability for flexible and efficient investigation of microprotein functions.
• Paved the way for discovering novel microprotein functions in developmental processes.

Conclusions:

• Microproteins play significant roles in biological processes and animal development.
• The CRISPR-Cas13d system is a powerful tool for studying microprotein functions, especially in contexts with maternally inherited transcripts.
• This approach facilitates the characterization of previously unannotated functional peptides during development.