CRISPR on/off
Over the past decade, the CRISPR-Cas9 gene editing system has revolutionized genetic engineering, allowing scientists to make controlled changes to the DNA of organisms. The system can potentially be used to treat various diseases, but editing involves cutting DNA strands, which leads to irreversible changes in the genetic material of the cell.
In an article published in the journal Cell, the researchers described a new gene editing technology CRISPRoff, which allows you to control the expression of genes with high specificity, leaving the DNA sequence intact. The method developed by Jonathan Weisman, an employee of the Whitehead Institute, Luke Gilbert, an associate professor at the University of California at San Francisco, James Nunez, a PhD candidate from Weisman's laboratory, and colleagues is stable enough to maintain the effect through hundreds of cell divisions, and is also completely reversible. This is a simple tool that can silence and turn on again any of the practical genes or several genes at the same time.
Genetic Engineering 2.0
The classic CRISPR-Cas9 system uses the DNA-cutting enzyme Cas9 found in bacteria. This system can target specific genes in human cells with a guide RNA that tells Cas9 proteins where to break the DNA chain. The DNA damage formed after editing is closed by the repair complex existing in the cell.
The classic CRISPR-Cas9 method changes the basic DNA sequence, therefore it gives a permanent and irreversible effect. In addition, dependence on one's own cellular repair mechanisms means that it will be difficult to limit oneself to one desired change and fully control the results.
Therefore, the researchers were looking for an opportunity for another type of gene editor, one that does not change the DNA sequence, but changes the way it is read in the cell.
Scientists call such modifications epigenetic – genes can be disabled or activated based on chemical changes in the DNA chain. Epigenetic silencing of genes often works through methylation – the addition of chemical labels to certain fragments in the DNA chain that make them inaccessible to the enzyme RNA polymerase, which reads genetic information from DNA into matrix RNA, with which, in turn, proteins are built. Weissman and colleagues had previously created two other epigenetic editors CRISPRi and CRISPRa, but both of them could only work in cells that constantly express artificial proteins that preserve epigenetic changes.
Creating a switch
To build an epigenetic CRISPRoff editor that could mimic natural DNA methylation, the researchers created an RNA-driven protein structure that can turn off target genes by attaching methyl groups to specific points on the DNA chain.
Since the new method does not alter the DNA sequence, researchers can reverse the silencing effect by using enzymes that remove methyl groups. They called this method CRISPRon.
By testing CRISPRoff under various conditions, the researchers discovered several interesting features of the new system. First, they were able to target it to the vast majority of genes in the human genome, and this worked not only for the genes themselves, but also for other regions of DNA that control gene expression but do not encode proteins.
In addition, CRISPRoff silenced genes that do not have CpG islands - regions previously considered necessary for any DNA methylation mechanism. Prior to this work, it was believed that 30% of genes that do not have a CpG island are not controlled by DNA methylation.
CRISPRoff in Research and Therapy
To explore the potential of CRISPRoff for practical application, scientists tested this method on induced pluripotent stem cells. These cells can transform into countless cell types in the body depending on the cocktail of molecules that surround them, and thus are powerful models for studying the development and functioning of certain cell types.
The researchers selected a gene that CRISPRoff turned off in stem cells, and then turned them into neurons. When they searched for the same gene in neurons, they found that it remained silent in 90% of cells, proving that cells retain memory of epigenetic modifications made by the CRISPRoff system, even when the cell type changes.
The researchers also showed how CRISPRoff can be used for therapy: they targeted a gene encoding a tau protein that is associated with Alzheimer's disease. The use of CRISPRoff reduced the expression of this protein in neurons, although it did not completely disable it.
But even if CRISPRoff is not able to cure Alzheimer's disease, there are many other conditions in which it can potentially be applied. While delivery to certain tissues remains a problem for gene editing technologies, including CRISPRoff, however, the authors have shown that it is possible to deliver it using vector systems.
CRISPRoff has great potential for research, enabling scientists to silence any part of the genome; it is a great tool for studying the function of certain genes.
In addition, having a reliable system for altering cell epigenetics will help researchers study the mechanisms by which epigenetic interventions are transmitted through cell divisions.
Article by J.K.Nuñez et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing is published in the journal Cell.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on the Whitehead Institute: An on-off switch for gene editing.