Toggling the epigenetic switches with CRISPR

Author: Kavya Subramanian
Artist: Qiwen Liu
Editor: Catherine Turnbull

CRISPR – whether you think it’s a bop or flop, you’ve probably heard of it at some point in the last few years! It could be because you’re an absolute academic weapon that read the Nobel-winning 2012 paper published by Jennifer Doudna and Emmanuelle Charpentier; it could be from the Netflix documentaries, the CRISPR patent war, or the designer babies; or it could be from listening to lecturers, or CRISPR enthusiasts like me. The point is that within a decade, CRISPR has become the popular kid on the biotech block.

‘CRISPR’ is simply an acronym, since ‘Clustered Regularly Interspaced Short Palindromic Repeats’ doesn’t quite roll off the tongue. In a nutshell, it is a genetic component of some bacteria’s antiviral defence, exploited by humans for genetic engineering. To preface the forthcoming subjects, simplifying some key concepts might be useful. DNA is like your aunt’s highly confidential and cryptically written soup recipe, while RNA is the working copy of that recipe – decoded and re-written in a way you understand. From this decoded recipe, you prepare the soup which is the protein or the enzyme of interest. However, your aunt’s recipe also has directions for her amazing bread to pair with the soup. The recipe now has two parts or ‘domains’ – the bread domain and the soup domain. Since you have some bread lying around, you decide not to follow the bread domain. But wouldn’t it be amazing if the bread domain could magically disappear and reappear into the recipe as and when you needed it instead of sifting through the recipe every single time? Biology does exactly that through ‘gene expression’ where selective DNA domains are decoded into RNA to only make necessary proteins, as and when needed.

CRISPR’s humble beginnings in genetic engineering was with the discovery of the Cas9 enzyme, which functions as a molecular scissor by binding to the DNA and snipping sections of it. A single stretch of RNA called single guide RNA (sgRNA) guides the Cas9 to scan the genome for regions of sequence complementarity. At these regions, Cas9 generates cuts called double stranded breaks (DSBs), and the cell uses its own DNA repair mechanisms to fix this break, generating a frameshift mutation in the process. The high programmability of the sgRNA to detect almost any genome sequence confers a great advantage to genetic engineering with CRISPR. Simply put, CRISPR-Cas9 is like the cut and paste mechanism to edit a cryptically written recipe.

However, this technology is not foolproof. CRISPR can sometimes generate off-target effects – a phenomenon that occurs when the genome is inadvertently cut at an unintended site. When used with Cas9, construction of the sgRNA must be incredibly specific to minimise these off-target effects. Additionally, frameshift mutations can silence genes, but to reactivate them, the reversal or changing of this mutation is far too complex. This limits CRISPR-Cas9 to modifying the DNA sequence and makes it an ineffective tool for modifying gene expression. Given the growing interest in epigenetics – the study of gene expression – and its predominant role in health and disease, the above-mentioned drawbacks majorly limit the therapeutic applications of CRISPR-Cas9. Consequently, members of the biotech community have been focussing on developing a tool for modifying and controlling gene expression, pushing the frontiers of what we now know as epigenome engineering.

CRISPR-based tools with modified or alternative versions of Cas9 are being discovered to overcome CRISPR-Cas9’s challenges. In 2021, researchers at the Whitehead Institute-MIT and the University of California, San Francisco jointly published a paper on a CRISPR-based epigenetic editor called CRISPRoff/on. This tool was made by fusing three domains, each coding for a modified dead Cas9 (dCas9), a transcriptional repressor called KRAB and an enzymatic epigenetic modifier. Transcriptional repressors are enzymes that slow down or stop the decoding of DNA to RNA – like your aunt’s newborn who keeps her busy and slows down her decoding the recipe for you. Enzymatic epigenetic modifiers are enzymes that indirectly modify gene expression. CRISPRoff contained an expression silencing methylase domain, and CRISPRon an expression activating demethylase domain. This technology is therefore a highly programmable epigenetic switch as the same tool can both ‘switch on’ and ‘switch off’ gene expression based on the epigenetic modifier domain. CRISPRoff/on is among the most exciting breakthroughs in epigenome editing, synergistically combining multiple epigenetic editing approaches from the past to produce heritable, reversible edits in a wide variety of cell types using a technology that is far more efficient and specific than its predecessors.

Increasingly, the therapeutic uses of the CRISPRoff/on system are being explored despite it not allowing for the dialling up or down of the silencing and activation levels. The above-mentioned scientists also discovered that the CRISPRoff/on system significantly, albeit not completely, silenced the tau gene in neurons. Misfolded tau proteins are found in the brain cells of Alzheimer’s patients, and the potential for gene silencing of erroneous tau using CRISPRoff could be applied therapeutically. Considering that the team that developed the CRISPRoff/on system comprised two of the co-founders of the U.S. based biotech company Chroma Medicine, it is unsurprising that they are actively further exploring the therapeutic role of epigenome editing. Among various investors in the biotechnology space, Tune Therapeutics has also been keen on exploring epigenome editing technologies. The two companies are now more hopeful than ever in merging epigenome editing with precision medicine.

These biotech companies believe that finding a suitable in vivo delivery mechanism for this technology is crucial in translating its effects therapeutically. Historically, the most commonly used gene delivery vectors (Adeno-Associated Viruses and Lipid Nanoparticles) have been ineffective in delivering CRISPRoff/on due to their own limitations. Active research on overcoming these challenges is directed at bringing an epigenetic angle to precision medicine. With gene therapy and RNA-based vaccines already becoming common therapeutic tools, it won’t be long before more researchers and biotechnologists find ways to harness the therapeutic benefits of integrating more genetics into healthcare. But until then, go make yourself some “good soup”!

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