CRISPR-Cas9 genome editing has revolutionized not only the way genomes are modified, but thanks to deactivated Cas9 (dCas9) and it’s growing arsenal of effector domains, it has also provided the tools needed for the epigenome editing revolution. However, to date, the tools to modify RNA itself have been lagging behind in the epigenome editing arms race. Thankfully, to keep the epitranscriptomic revolution going, two new studies report some much needed tools for editing N6-methyladenosine (m6A) RNA methylation.
m6A modification of RNA is a regulator of nearly all post-translation processes. This modification is dynamic with writer, eraser, and reader proteins much like DNA methylation and histone modification. Heterodimers of METTL3 and METTL14 carry out the catalytic and RNA-binding functions of the writer complex while ALKBH5 or FTO erase m6A. Exactly how m6A regulates RNAs is still unclear. However, specific proteins read m6A, including YTHDF1 which promotes translation efficiency.
Beyond CRIPSR: CIRTS Gets the Job Done at Half the Size
Targeting of CRISPR-Cas9 complexes to RNA has been accomplished already using catalytically inactive dead Cas13 (dCas13), which is a Cas9 related protein family that natively targets RNA. This approach has been used to image RNA with GFP and to alter splicing[BL1] . A problem with the dCas9 and dCas13 proteins is their large size. This presents challenges for viral packaging and direct protein delivery. Furthermore, many people already have circulating antibodies to these proteins. To combat these challenges, the lab of Bryan Dickinson at the University of Chicago sough out to engineer a new system that broke up the functions of CRISPR-Cas13 into modular protein components. Based on all the functions of Cas13 proteins, they knew they would need four components: an RNA hairpin-binding protein as the core of the system, a guide RNA (gRNA), a charged protein that could bind to the displayed gRNA sequence non-specifically to stabilize the gRNA prior to target engagement, and an effector protein of interest. They developed CRISPR-Cas-inspired RNA targeting system (CIRTS) to meet these challenges. Here are their findings:
- They found a set of viral-origin effector
proteins, RNA hairpin binding proteins, and ssRNA binding proteins that work effectively in vitro
- Swapping out these proteins to produce 10 different CIRTS combinations can be used to achieve diverse functions
- A humanized version also works effectively
- CIRTS can be delivered to living cells with AAV viral vectors
- They were able to knockdown protein production and trigger increased protein production with a YTHDF1 effector protein
- Multiple RNAs could be targeted orthogonally using the same effector module but different hairpin-binding modules
CIRTS is less than half the size of current RNA-targeting technologies, which means that it offers major research and translational opportunities.[BL2] [EC3] The authors hope that researchers will be able to use their platform to better understand m6A biology as well as develop better targeted therapeutics.
Cas9 Can Target Writing and Erasing RNA modifications
The precise mechanism by which m6A regulates RNA is unclear. The modification peaks in 5’UTRs and stop codons, but understanding the contribution of specific sites is hindered by the lack of high-resolution techniques to modify m6A. Current methods rely on modifying the adenine base itself, which can have unwanted effects and complicate the data. The lab of Shu-Bing Qian at Cornell University was looking tackle this problem and develop a system to write and erase specific m6A modifications without affecting the RNA sequence in human cell lines. Here’s what happened:[BL4]
- They engineered METTL3 and METTL14 into a single polypeptide chain with the dCas9 protein, which can effectively deliver m6A to gRNA targets
- m6A can target specific single sites in 5’UTRs
that are sufficient to induce non-canonical translation
- Induction of m6A in the 3’UTR modifies mRNA turnover, but is not site-specific
- To create targetable m6A erasers, the authors
fused either ALKBH5 or FTO to dCas9
- Both can target specific nucleotides on mRNAs of interest for m6A erasure
Possible further applications of this technology include altering RNA secondary structure without altering primary sequence. The authors hope their technology will serve as a platform for developing toolkits to edit the diverse base and sugar modifications to RNA.
Together, these two studies hold promise for further adding to the already impressive epigenome editing arsenal. Modulation of RNA levels and alteration of RNA modifications add another method of regulating gene expression, and thus cellular function, which may be preferable to genome editing in many cases.