Mapping the dark matter of the genome is no easy chore. Enhancers love to hide and often don’t act as expected. But thankfully, there’s a handy new CRISPR/Cas9 tool to fluoresce the genomes darkest regulatory secrets. The multiplexed editing regulatory assay (MERA) is a clever system that pushes back the limits of CRISPR/Cas9 as a genome-wide screening tool.
MERA was created in mouse embryonic stem cells (mESCs) by using three rounds of electroporation to knock in the desired components via CRISR/Cas9-mediated homologous recombination. It also introduced a clever new trick for homologous recombination that involved using PCR to create 79-90 bp homology arms around the sequence to be inserted.
Here’s how the talented team from MIT & Harvard did it:
- In the first round of CRISPR, a construct for a single sgRNA that produced a dummy hairpin was knocked into the universally accepting ROSA26 locus.
- In the second round of CRISPR, homologous recombination was used to knock in GFP at a gene of choice (Nanog, Rpp25, Tdgf1 or Zfp42).
- In the third round of CRISPR, a sgRNA library was delivered that tiled thousands of mutations on a ~40kb region around the GFP-gene fusion that was suspected of hiding enhancers.
- This approach allowed the team to swap in one sgRNA per cell at the eagerly awaiting dummy site.
- Next, with all the CRISPRing out of the way and the enhancer tiling sgRNA library randomly distributed, they used flow cytometry to sort the cells according to their fluorescence.
- Finally, the sorted cells were deep sequenced to determine which sgRNAs decreased GFP expression and thus hit a regulatory ‘sweet spot’.
Using MERA, they found not only proximal and distal regulatory elements but also ‘funky’ unmarked regulatory elements (UREs) that weren’t associated with the expected DNase hypersensitivity or histone modifications. While promising, the MERA system does have some limitations, since it won’t work for in vivo screens or cell lines that aren’t fans of homologous recombination. However, despite these limitations, MERA demonstrates the raw power of CRISPR when it comes to uncovering new classes of enhancers and fine-mapping functional motifs.
Go take in all the CRISPR that MERA offers over at Nature Biotechnology, January 2016