Ever since its discovery, CRISPR research has made advances in leaps and bounds to edit the genetic code. There have been many variations to the core technology that enable the targeting of specific DNA regions in the genome through a guide RNA and editing it using the Cas9 enzyme.
The scientific community is striving to cure numerous genetic diseases with this tool. However, there is extreme caution in employing this Nobel Prize-winning tech to edit germ cells since DNA repair is a complex process and CRISPR could lead to unintended consequences with off-target editing.
Evading Off-Target Effects via Reversible CRISPR
In a new study published in Cell, scientists from MIT and UCSF describe a new CRISPR tool—CRISPRoff, which can turn off genes without making changes to their genetic code. This switch is heritable and stably passed on to subsequent generations. Besides, it can be reversed by a complementary tool—CRISPRon.
Employing epigenetic marks, CRISPRoff can silence a broad spectrum of genes, even those without CpG islands with high specificity. This switch-off is maintained even when cells differentiate into specialized lineages.
The CRISPR on-off technology provides some major advantages over conventional therapy in that the DNA code is unedited, so there are no unwanted off-target effects. It is a reversible tool, so the genes can be turned back on when needed. The design of the CRISPRoff is similar to conventional CRISPR in the guide RNA design.
However, the Cas9 enzyme is replaced by DNA methylase, which adds methyl groups to the target DNA. Methylation is a common feature of gene silencing. The design included proteolysis-resistant linkers to ensure that the methylase does not detach from the guide RNA and cause off-target methylation.
Strikingly, these methyl tags can be removed by a similar CRISPRon, which performed targeted demethylation and restored gene expression. The authors demonstrated the efficacy and specificity of this epigenetic gene editing in a wide variety of cell lines- HEK293T, HeLa, U2OS, and stem cells. The CRISPRoff tool could also be applied to silence multiple genes simultaneously without DNA damage.
The CRISPRoff technology is particularly attractive as a therapeutic tool for its reversibility and ability to silence genes without CpG islands. Conventional wisdom in the epigenetic field is that genes without CpG islands (regions of DNA with a high concentration of CG bases) cannot be turned off by methylation, which is about 30% of our genome. However, in this study, the CRISPRoff could silence genes with or without the CpG islands, which is quite a paradigm shift in our understanding of epigenetics.
The editing by CRISPRoff was seen even in the corresponding differentiated cells when the editing was done in stem cells, suggesting that epigenome editing is faithfully maintained during cell differentiation and maturation. This makes CRISPRoff an ideal tool to develop for therapy of genetic disorders where a single jab can silence the disease-associated gene, much like the Covid vaccines available today.
The authors demonstrated a proof-of-concept with silencing Tau protein, whose overexpression and accumulation in the brain causes Alzheimer’s disease. However, it needs more work to improve tissue-specific delivery and optimal transient delivery before it is a viable form of therapy.
The ability to turn on/off genes can greatly enhance our ability to study epigenetic marks, their effects on gene expression, the mechanism of their heritability. This can be made possible by the novel CRISPRoff technology, which can further our understanding of the complex interplay between genome and epigenome.
In a press release, one of the lead authors, Prof. Luke Gilbert, said, “With this new CRISPRoff technology, you can [express a protein briefly] to write a program that’s remembered and carried out indefinitely by the cell. It changes the game, so now you’re basically writing a change that is passed down through cell divisions — in some ways, we can learn to create a version 2.0 of CRISPR-Cas9 that is safer and just as effective and can do all these other things as well.”
Editor: Rajaneesh K. Gopinath, Ph.D.
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