For the last few years, DNA has cornered the methylation market as the Apple of epigenome editing tools, but a newly developed CRISPR-Cas13 system has epigenetic investors buying stock in RNA editing!
6-methyladenosine (m6A) is the most abundant RNA modification, and even though we know it’s involved in chromatin accessibility, RNA splicing and repairing damaged DNA, scientists still struggle to understand m6A’s exact share in the epigenetic market. Before this breakthrough researchers had to invest their funds in knocking down or overexpressing the entire RNA methylation landscape, now but members of David Liu’s lab (Harvard University, Boston, USA) have developed a new targeted RNA methylation tool that allows them to diversify.
Introducing dCas13-M3: The Targeted m6A Methylator
The talented team used a catalytically inactive Cas13 protein (dCas13), which binds to single stranded RNA, to find that:
- The METTL3 methyltransferase is a great effector domain when you remove the zinc finger domain, since that creates the right balance between effectively transferring radiolabelled methyl groups to an RNA substrate, and reducing its natural RNA binding affinity
- Attaching this mutant protein to the C terminus of dCas13 (referred to as dCas13-M3), and adding it to E. coli with guide RNA (gRNA) increases the methylation of a synthetic RNA construct, as measured by MeRIP-qPCR and immunofluorescence
- dCas13-M3 also adds 2-3 times more methylation to a synthetic substrate in mammalian HEK293T cells compared to a non-specific control
To maximize their scientific dividends, the brainy epigenetic brokers rearranged the components of their system and added nuclear localisation (NLS) or export signals (NES) and/or METTL14 (M14), the endogenous binding partner of METTL3, and found:
- The proteins localize to their expected cellular compartment in HEK293T cells, even when they are targeted to a transcript that normally shuttles proteins between the nucleus and the cytoplasm
- Both the nuclear and cytoplasmic constructs increase methylation at the 3’UTR of transcripts, even when they are already endogenously methylated in HEK293T cells
- The most active constructs are dCas13-M3nls and dCas13-M3M14nes, and both systems install maximal methylation when gRNAs are designed to bind 30bps upstream, and end 8-15bp away from the target adenine
- dCas13-M3nls has no effect on global levels of m6A, when measured using immunofluorescence, but dCas13-M3M14nes increases background methylation by 1.3-fold
- dCas13-M3M14nes also generates 6 times more off-target MeRIP-seq peaks than dCas13-M3nls, although both have a substantial effect on transcription when transfected without specific gRNAs
Not sold yet? Compared to the dCas9-based system that’s already on the market, dCas13-M3nls works without needing an additional PAM oligionucleotide, and has fewer off target MeRIP-seq peaks (2.3% compared to 15.4%), and can still decrease target mRNA levels or induce alternative splicing of its target!
Buy low, sell high, and capitalize on all the details in the original article in Nature Biotechnology, June 2020.