NEW YORK – Researchers have developed a CRISPR-based system using the Cas13 enzyme capable of knocking down maternal and zygotic mRNA in zebrafish embryos in a systematic and precise way, in order to be able to recapitulate developmental phenotypes and interrogate gene functions in the embryos.
In a paper published on Friday in Developmental Cell, an international team of researchers said that although maternal genes are critical for establishing early developmental programs, the study of their functions in embryos has been challenging due to a lack of available techniques for their systematic disruption and assessment. CRISPR-Cas13 platforms have been used to degrade RNA in yeast, plants, and mammalian cell lines, but haven't been systematically studied in animal systems.
In their study, the researchers developed a platform using a Cas13 ortholog called RfxCas13d as an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos, and used it to target zygotically expressed and maternally provided transcripts, resulting in a 76 percent average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. They also demonstrated that the system can be used in medaka, killifish, and mouse embryos.
"The exciting thing about this study is not just what we found, but what we can do," co-senior author Ariel Bazzini, an assistant investigator at the Stowers Institute for Medical Research, said in a statement. "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. … That maternal contribution [to the embryo] is a mystery that many of us want to solve."
By using Cas13 instead of Cas9, the researchers developed a platform that targets RNA rather than DNA. By directly manipulating RNA activity in a relatively tunable manner, they can study how subtle changes in transcript levels affect biological processes. Further, by reducing but not completely removing gene activity, they can uncover phenotypes that may otherwise remain hidden due to loss-of-function lethality. And therapeutic approaches that target RNA can also have an advantage over those that target DNA in that they provide temporary modifications without the prospect of permanent heritable changes.
The researchers began by testing four Cas13 orthologs. The mRNA for each Cas13 variant was injected individually into one-cell stage zebrafish embryos, and they found that RfxCas13d efficiently disrupted maternal and zygotic gene function without embryonic toxicity and with specificity in zebrafish embryos. Notably, Cas13d protein injection accelerates maternal RNA depletion and consequently provides more penetrant phenotypes.
To address whether the CRISPR-RfxCas13d system could be used to target maternally deposited mRNAs, the researchers injected zebrafish embryos derived from a transgenic mother that expresses the rfp mRNA at very high levels with RfxCas13d mRNA (mCas13d), gfp mRNA, plus three guide RNAs (gRNA) targeting rfp. Fluorescence intensity was visibly reduced when compared with embryos injected with mCas13d or gRNAs alone, or by co-injection of mCas13d with unrelated gRNAs, the researchers found. Further, rfp mRNA levels were reduced approximately 4.4-fold when compared with embryos injected with mCas13d alone.
Importantly, the researchers said, within the co-injected embryos with mCas13d and gRNAs targeting rfp mRNA, rfp was the most significantly downregulated transcript, indicating that the CRISPR-RfxCas13d system can functionally disrupt the activity of maternally provided mRNAs in a specific manner during early development in zebrafish embryos.
They next set out to assess the efficacy and specificity of targeted gene knockdown on both maternally provided and/or zygotically expressed mRNAs in zebrafish embryos. To assess transcriptome-wide effects after endogenous gene knockdown, the investigators targeted the szrd1 mRNA. They saw a sevenfold reduction in levels of szrd1 mRNA expression in embryos injected with the CRISPR-RfxCas13d platform compared with embryos injected with mCas13d alone, and saw no other substantial mRNA changes.
They also tested the CRISPR-RfxCas13d method on endogenous mRNAs with known roles in embryonic development and previously characterized loss-of-function phenotypes. For example, the researchers targeted dnd1 mRNA, whose impairment abolishes primordial germ cell migration and survival. Embryos co-injected with mCas13d and three gRNAs targeting dnd1 were devoid of detectable germ cells, as indicated by loss of germ-cell-specific GFP expression. In contrast, germ cells were not visibly affected by injection of mCas13d alone or in conjunction with gRNAs targeting an unrelated mRNA.
Taken together, these results suggest that the CRISPR-RfxCas13d system is specific, the researchers said. Further experiments also demonstrated that CRISPR-RfxCas13d efficiently induced maternal and zygotic mRNA knockdown, recapitulated early embryogenesis phenotypes in zebrafish embryos, and was associated with minimal toxicity, off-targeting, and stress or immune response activation.
"We think this tool could have a profound effect on our understanding of infertility and developmental problems in general," Bazzini noted in his statement.
The authors also concluded that the techniques described in the study could act as a jumping off point for the implementation of other CRISPR-Cas13 applications in vivo, such as RNA editing, transcript tracking, and imaging optimized in mammalian cells.