DNA methylation got your expression down? Why not try targeted demethylation with Casilio-ME? CRISPR/Cas9 techniques have been applied to numerous biological tasks including regulation of DNA methylation. 5-methyl cytosine (5mC) is deposited by DNA methyltransferases (DNMTs) and removed by step-wise oxidation via the three ten-eleven translocation (TET) enzymes; 5mC is first converted to 5-hydroxymethylcytosine (5hmC), then 5-formylcytosine (5fC), then 5-carboxylcytosine (5caC). 5fC and 5caC are then removed by the base-excision repair (BER) machinery. Tethering of TET1 or DNMT3a to deactivated Cas9 (dCas9) proteins has been used as a method to regulate 5mC at specific sites; however, dCas9 is limited in its ability to enable multiplexed targeting, effector multimerization, or formation of protein complexes at the targeted sequence. This means that the multiple proteins involved in DNA methylation and demethylation cannot be easily guided to specific sites using traditional dCas9 methods.
The lab of Albert Cheng at The Jackson Laboratory (USA) set out to develop a DNA Methylation Editing platform. They wanted to link TET1 activity to other demethylation machinery to efficiently alter the epigenetic state of CpG targets and activate silenced genes. The previously developed a technique they call “Casilio” which uses a nuclease-deficient Cas9 (dCas9), an effector module made of Pumilio/FBF (PUF) domain linked to an effector protein, and a modified sgRNA containing PUF-binding sites (PBS). Multiple PBS added in tandem to the 3′ end of the sgRNA allow concurrent recruitment of multiple PUF-effectors to targeted DNA sequences. Here, they created three versions of this technique and tested them out in human cell lines. Here’s what they did:
- The first iteration, Casilio-ME1, was much more efficient at activating 5mC-repressed genes than previously developed targeted TET1 delivery systems (SunTag, TALEs and MS2)
- Casilio-ME2 guides both TET1 and GADD45A (believed to couple 5mC oxidation to DNA repair) to target sites, resulting in a 3-6 fold increase in target gene expression over Casilio-ME1 (TET1 alone)
- They also developed Casilio-ME3, TET1 plus the BER enzyme NEIL2 which resulted in a 4-fold increase in target activation over Casilio-ME1
- They did not find any off-target effects using reduced representation bisulfite sequencing, but did identity several off-target genes differentially expressed genes by RNA-seq
The authors hope that this platform can be used and further developed to activate 5mC-silenced genes to desired expression levels. Also, it could be applied to test the causal role of 5mC in silencing genes of interest in specific contexts.
Go activate your genes with the full-length story in Nature Communications, Sept 2019