Enhancers are hugely important regulatory regions of DNA that control gene expression. New techniques are telling us a lot about enhancer structure and function, but the study of enhancers could still use some enhancement. Enhancers are currently defined by what they look like (local DNA methylation, histone modifications, and chromatin availability) or by pulling them out and testing their ability to activate the expression of reporter genes in an artificial context. This is great, but both approaches still only circle around how the enhancer actually works in its native context.
Scientists can make small deletions in enhancers to test candidate regions within them, but this approach has been too low throughput to provide a detailed picture. Now, using CRISPR/Cas9, a team at Harvard has shown how to functionally map an enhancer to near-base pair resolution.
Functional Mapping of the BCL11A Enhancer
The enhancer in question was for BCL11A, a key player in many inherited hemoglobin disorders. To map this enhancer in situ, the team created a library of all possible short guide RNAs (sgRNAs) targeting the enhancer and cloned it into a lentiviral vector. Since they were using Cas9 from S. pyrogenes, they were limited to target sequences adjacent to the NGG PAM sequence, but they still had excellent coverage of the enhancer.
Since the lentiviral vector did not include a template for homology directed repair (HDR), the CRISPR targeting induced small indels from non-homologous end joining (NHEJ). In gene editing, NHEJ can be a problem, but in this case the small mutations were just what the team needed for fine mapping.
Next, they transfected this library into a human cell line and sorted cells by expression of fetal hemoglobin HbF, which would increase if the enhancer had been disrupted. Finally, they sequenced the cells to see which sgRNAs they had incorporated, and then calculated an enrichment score comparing each sgRNA frequency in high HbF (disrupted enhancer) cells vs in low HbF cells. This revealed that:
- sgRNA enrichment localized to a few specific clusters.
- These clusters overlapped with expected chromatin features and transcription factor binding motifs.
- Enhancers could be mapped to even finer detail by sorting and sequencing cells exposed to specific sgRNAs to see which exact indels changed expression.
- Mouse experiments supported a key role for the enhancer elements in a developmental switch from fetal haemoglobin to the adult form.
This work is an excellent use of CRISPR for fine mapping of enhancers, and it identified an exact locus as a potential target for gene therapy in the future. As the authors note, one could potentially reach even higher resolution by using multiple Cas9 proteins with different PAMs to get even denser arrays of sgRNAs.
So if you’ve been feeling lost lately, enhance your own mapping with this paper by co-first authors Matthew Canver, Elenoe Smith, Falak Sher, Luca Pinello, and Neville Sanjana and co-supervisors Feng Zhang, Stuart Orkin, and Daniel Bauer, over at Nature, 2015.