CRISPR Used to Knock Down Maternal and Zygotic mRNA and Study Gene Messages in Early Development

CRISPR Used to Knock Down Maternal and Zygotic mRNA and Study Gene Messages in Early Development

Embryonic development starts when eggs are fertilized by sperm, and the resultant embryonic genome has half of its genes provided by the father and half from the mother, but the eggs also have proteins and RNA that are provided by the mother that kickstart the developmental process.

Studying maternal gene functions has proven difficult, as while it is possible to use CRISPR and other gene editing techniques to manipulate genes, it has not been possible to knock out some genes because they are essential to life and current technologies do not support systematic gene disruption and assessment. The RNA-interference method that is used to study gene function does not work in zebrafish embryos, which are the preferred choice for developmental biologists. RNA interference also doesn’t work in other fish or frog embryos.

An international team of researchers from the Stowers Institute for Medical Research in Kansas City, Missouri, and the Andalusian Center of Developmental Biology at Pablo de Olavide University in Seville, Spain, have made a breakthrough and have identified a Cas13 enzyme that has allowed them to knock down maternal and zygotic mRNA accurately, with minimal toxicity and off-target effects.

The researchers tested four different Cas13 proteins but found that three were either toxic to zebrafish embryos or were inefficient. The fourth was a Cas13 ortholog called RfxCas13d that was not toxic and allowed them to efficiently target maternal and zygotic messenger RNA (mRNA) and deplete specific mRNA transcripts in zebrafish embryos. The system can also be used in killifish medaka, and mouse embryos.

The new method has allowed the researchers to study subtle changes in transcript levels to see how they affect biological processes. The platform developed by the researchers is tunable and has allowed them to reduce the average transcript levels by 76% and recapitulate embryonic phenotypes.

“The exciting thing about this study is not just what we found, but what we can do,” said Ariel Bazzini, Ph.D., an assistant investigator at the Stowers Institute and co-lead author of the study. “We still don’t understand how genes jumpstart the earliest stages of development. Now we can find out by targeting their RNA messages, one by one.”

The method developed to manipulate RNA is low cost and relatively easy to perform. The researchers have already distributed the necessary reagents and the method to several labs around the world. The researchers say any lab currently working with zebrafish and other animal embryos could easily adopt the new technique.

“We think this tool could have a profound effect on our understanding of infertility and developmental problems in general,” said Bazzini.

You can read more about the study in the paper – CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos – which was recently published in the journal Developmental Cell. DOI: 10.1016/j.devcel.2020.07.013

Leave a Reply