Epigenome Editing with CRISPR-dCas9, TALEs, and Zinc Fingers
With a reference epigenome on the horizon it has become apparent that an enormous amount of variation, both inherited and environmental, is responsible for the development of complex traits and diseases. While a catalogue of such alterations is invaluable to humanity, perhaps more valuable would be the ability to alter and correct these modifications. Genome editing systems have quickly risen to popularity for their ability to precisely manipulate sequence at will. However, these biotechnologies have come in another flavour; one that is meant for the epigenome.
Epigenome editing shares many similarities with genome editing, as they are both based on the same core biotechnology. Genome editing utilizes two components: a targeting system and a nuclease. Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) are early genome editing technologies modified from biological systems that are meant to regulate gene expression rather than modify sequence. During genome editing both ZFNs and TALENs utilize a nuclease (FokI) that requires dimerization in order to cut. They also rely on protein-DNA interactions for their sequence specificity, which is relatively costly and time consuming to produce due to the required protein engineering. Additionally, specificity is also influenced by local chromatin modifications. On the other hand, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system associated proteins, like Cas9 and Cpf1, are nucleases from a larger adaptive immune system in bacteria and archaea. CRISPR-Cas9 differs from earlier genome editing technologies as it uses a short synthetic guide RNA (sgRNA) for sequence specificity, which allows for an accelerated editing timeline. Additionally, the system appears to work across much more of the tree of life. Genome editing can be used to edit sequence critical to the epigenome, which is distinct from epigenome editing, and is referred to as genome editing the epigenome. This strategy involves targeting sites critical for chromosome conformation or transcription factor binding. As the majority of genome wide association study (GWAS) variants lie in regions outside protein coding sequence they are prime targets for genome editing.
However, it is also possible to use effector domains to manipulate epigenetic marks without altering the underlying sequence! This is known as epigenome editing. While the earlier systems swap their added nuclease for epigenetic effector domains to enable novel functions, Cas9 can have its nuclease activity precisely deactivated, which is known as dCas9. Despite being deactivated dCas9 can be fused to various effector domains and still utilize the sgRNA for targeting.
Epigenome editing came about from the desire to create artificial transcription factors that could be used for transcriptional activation, also known as transactivation, or transcriptional repression. Interestingly, some artificial transcription factors would also leave behind altered epigenetic marks, either from directly recruiting them or as indirectly as a consequence of altered transcriptional activity in the targeted region. However, epigenome editing arose in 2002 from Snowden et al., whose group used G9a (aka EHMT2 in humans) as an effector domain with a custom Zinc Finger to show that H3K9 causes transcriptional repression in vivo. Epigenome editing systems have been created to utilize effector domains ranging from a variety of histone modifying proteins, DNA methyltransferases and TET enzymes, as well as a number of non-coding RNAs. Alternatively, effector domains can be used to immunoprecipitate or visualize desired sequence. Even more regulatory abilities can be added by combining epigenome editing systems with other biotechnologies, such as using optogenetics to allow for inducibility. Finally, designer improvements have also been made, which involve genetically engineering the system in order to greatly increase the strength of transactivation and allow for the studies of enhancers and gene families.
Just like genome editing systems, epigenome editing systems aren’t without their challenges. Some shared challenges include delivery of the system to host cells, off-target effects, cytotoxicity, and the ethics of certain applications. One challenge unique to epigenome editing is that sometimes the modifications are short lived and multiple modifications or different regions may need to be targeted to achieve a long-term effect. However, given the rapid rate of growth of these particular biotechnologies the aforementioned obstacles will shortly be overcome. No longer is the ability to manipulate the epigenome limited to labs specialized in genetic engineering.
Laufer BI, & Singh SM (2015). Strategies for precision modulation of gene expression by epigenome editing: an overview. Epigenetics & chromatin, 8 PMID: 26388942