A study published today in Developmental Cell establishes the use of CRISPR-Cas technology to target RNA in embryonic animal models in a specific and systematic manner.
By disrupting gene messages (RNA) instead of the underlying genes (DNA), researchers can study genes that might previously have been difficult or impossible to manipulate because they were essential to life or involved in a critical stage of biological development. This approach also allows targeting of maternally-contributed gene RNAs, which are deposited in the egg to kick off the earliest genetic programs.
"That maternal contribution is a mystery that many of us want to solve," says Ariel Bazzini, Ph.D., an assistant investigator at the Stowers Institute and co-leader of the study. However, attempts to systematically target RNA in zebrafish have been unsuccessful. So when the team saw reports that CRISPR technology had been employed to degrade RNA in yeast, plants, and mammalian cells, they were eager to give it a try. The researchers tested four different Cas13 proteins that had been successfully used in previous studies. They found that the Cas13 proteins were either inefficient or toxic to the developing zebrafish, except for one protein, called RfxCas13d.
They then examined whether targeting RNA with CRISPR-RfxCas13d in zebrafish embryos could recreate the same defects as altering the organism's underlying DNA. For example, when they targeted the RNA of the tbxta gene, which is necessary for growing a tail, the zebrafish embryos were tailless.
The researchers went on to show that the CRISPR system could efficiently target a variety of RNAs, both those provided by the mother as well as those produced by the embryo, decreasing RNA levels by an average of 76%.
"The CRISPR-RfxCas13d system is an efficient, specific and inexpensive method that can be used in animal embryos in a comprehensive manner," says Miguel A. Moreno-Mateos, Ph.D., who is also co-leader of the study. "With this tool we will help to understand fundamental questions in biology and biomedicine."