Scientists led by a team at the Whitehead Institute have developed a new gene editing technology, called CRISPRoff, which can be used to control gene expression with high specificity, while leaving the sequence of the DNA unchanged. Utilizing a programmable, epigenetic memory writer protein to silence gene expression, CRISPRoff is a highly specific method that effectively programs epigenetic memory that is heritable for hundreds of cell divisions. Designed by Whitehead Institute Member Jonathan Weissman, PhD, University of California San Francisco assistant professor Luke Gilbert, PhD, Weissman lab postdoc James Nu\u00f1ez, PhD, and collaborators, the method is also fully reversible.<\/p>\n\n\n\n
Reporting on the technology in Cell, the developers of CRISORoff, headed by Whitehead Institute Member Jonathan Weissman, PhD, demonstrated that CRISPRoff epigenetic memory persisted through differentiation of human induced pluripotent stem cells (hiPSCs) into neurons. \u201cThe big story here is we now have a simple tool that can silence the vast majority of genes,\u201d said Weissman, who is also a professor of biology at MIT and an investigator with the Howard Hughes Medical Institute. \u201cWe can do this for multiple genes at the same time without any DNA damage, with great deal of homogeneity, and in a way that can be reversed. It\u2019s a great tool for controlling gene expression.\u201d<\/p>\n\n\n\n
Weissman and co-developers Luke Gilbert, PhD, an assistant professor at the University of California San Francisco, Weissman lab postdoc James Nu\u00f1ez, PhD, and collaborators, describe the development of CRISPRoff in a paper titled, \u201cGenome-wide programmable transcriptional memory by CRISPR-based epigenome editing.\u201d<\/p>\n\n\n\n
The project was partially funded by a 2017 grant from the Defense Advanced Research Projects Agency to create a reversible gene editor. \u201cFast forward four years [from the initial grant], and CRISPRoff finally works as envisioned in a science fiction way,\u201d said co-senior author Gilbert. \u201cIt\u2019s exciting to see it work so well in practice.\u201d<\/p>\n\n\n\n
The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9, which is found in bacterial immune systems. Using a single guide RNA, the system can be targeted to specific genes in human cells, where the Cas9 proteins create tiny breaks in the DNA strand. The cell\u2019s existing repair machinery then patches up the holes.<\/p>\n\n\n\n
Because these methods alter the underlying DNA sequence, they are permanent. Plus, their reliance on \u201cin-house\u201d cellular repair mechanisms means it is hard to limit the outcome to a single desired change. \u201cThese technologies have been optimized for targeted changes in the underlying DNA sequence and are therefore ideally suited for repairing or introducing pathogenic mutations,\u201d the authors explained. \u201cHowever, the reliance on endogenous DNA repair machinery presents challenges, because the complexity of these pathways can make it difficult to limit the outcome to a single desired change.\u201d Weissman noted, \u201cAs beautiful as CRISPR-Cas9 is, it hands off the repair to natural cellular processes, which are complex and multifaceted,\u201d Weissman says. \u201cIt\u2019s very hard to control the outcomes.\u201d<\/p>\n\n\n\n
The team realized an opportunity to develop a different kind of gene editor that didn\u2019t alter the DNA sequences themselves, but instead changed the way they were read in the cell. \u201cAn alternative modality for modulating gene function is to rewrite the epigenetic landscape to control gene expression without changing the underlying DNA sequence,\u201d they wrote. This epigenetic strategy is designed to silence or activate genes based on chemical changes to the DNA strand. Problems with a cell\u2019s epigenetics are responsible for many human diseases such as Fragile X syndrome and various cancers, and can be passed down through generations.<\/p>\n\n\n\n
Epigenetic gene silencing can work through methylation\u2014the addition of chemical tags to certain places in the DNA strand\u2014which causes the DNA to become inaccessible to the RNA polymerase enzyme that reads the genetic information in the DNA sequence into the messenger RNA transcripts, which can ultimately represent the blueprints for proteins.<\/p>\n\n\n\n
Weissman and collaborators had previously created two other epigenetic editors called CRISPRi and CRISPRa\u2014but both of these came with a caveat. In order for them to work in cells, the cells had to be continually expressing artificial proteins to maintain the changes. \u201c\u2026 current programmable epigenome editing technologies typically rely on constitutive expression of Cas9-fusion proteins to maintain transcriptional control,\u201d the team noted. \u201cAs such, these modalities remain less suitable for therapeutic cell and organismal engineering.\u201d<\/p>\n\n\n\n
To build an epigenetic editor that could mimic natural DNA methylation, the researchers created a tiny protein machine that, guided by small RNAs, can tack methyl groups onto specific spots on the strand. These methylated genes are then silenced, or turned off, hence the name CRISPRoff. Because the method does not alter the sequence of the DNA strand, the researchers can reverse the silencing effect through the use of enzymes that remove the methyl groups, a method that they called CRISPRon.<\/p>\n\n\n\n
\u201cWith this new CRISPRoff technology, you can [express a protein briefly] to write a program that\u2019s remembered and carried out indefinitely by the cell,\u201d said Gilbert. \u201cIt changes the game so now you\u2019re basically writing a change that is passed down through cell divisions\u2014in 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.\u201d<\/p>\n\n\n\n
As they tested CRISPRoff in different conditions, the researchers discovered that they could target the method to the vast majority of genes in the human genome, and that also it worked not just for the genes themselves, but for other regions of DNA that control gene expression but do not code for proteins. \u201cOur initial experiments demonstrate CRISPRoff can perturb enhancers, opening the potential to target genome elements that control tissue-specific gene expression.\u201d First author Nu\u00f1ez acknowledged, \u201cThat was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes.\u201d<\/p>\n\n\n\n
In addition, and again surprisingly, CRISPRoff was able to silence genes that did not have large methylated regions called CpG islands (CGIs), which had previously been thought necessary to any DNA methylation mechanism. \u201cWhat was thought before this work was that the 30% of genes that do not have a CpG island were not controlled by DNA methylation,\u201d Gilbert commented. \u201cBut our work clearly shows that you don\u2019t require a CpG island to turn genes off by methylation. That, to me, was a major surprise.\u201d<\/p>\n\n\n\n
To investigate the feasibility of applying CRISPRoff for practical applications, the scientists tested the method in induced pluripotent stem cells, which represent useful models for studying the development and function of particular cell types. The researchers chose a gene to silence in the stem cells, and then induced the stem cells them to differentiate into neurons. Encouragingly, the CRISPRoff-targeted gene remained silenced in 90% of the resulting stem cell-derived neurons, revealing that cells retain a memory of epigenetic modifications made by the CRISPRoff system even as they change cell type.<\/p>\n\n\n\n
The researchers also selected the gene that codes for the Tau protein\u2014which is implicated in Alzheimer\u2019s disease\u2014to use as an example of how CRISPRoff might be applied to therapeutics. After testing the method in neurons, they were able to show that CRISPRoff could be used to turn Tau expression down, although not entirely off. \u201cWhat we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed,\u201d Weissman said. \u201cThe question is, then, how do you deliver this to an adult? And would it really be enough to impact Alzheimer\u2019s? Those are big open questions, especially the latter.\u201d<\/p>\n\n\n\n
Even if CRISPRoff does not lead to Alzheimer\u2019s therapies, there are many other conditions it could potentially be applied to, the researchers suggest. And while delivery to specific tissues remains a challenge for gene editing technologies such as CRISPRoff, as Weissman noted, \u201cwe showed that you can deliver it transiently as a DNA or as an RNA, the same technology that\u2019s the basis of the Moderna and BioNTech coronavirus vaccine.\u201d<\/p>\n\n\n\n
The scientists are enthusiastic about the potential of CRISPRoff for research as well. \u201cSince we now can sort of silence any part of the genome that we want, it\u2019s a great tool for exploring the function of the genome,\u201d Weissman noted. \u201cThe broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance,\u201d the authors concluded.<\/p>\n\n\n\n
The availability of a reliable system to alter a cell\u2019s epigenetics could in addition help researchers learn more about the mechanisms by which epigenetic modifications are passed down through cell divisions. As the authors stated, \u201cMore generally, this system allows us to broadly explore the biological rules underlying epigenetic silencing and provides a robust tool for controlling gene expression, targeting enhancers, and exploring the principles of epigenetic inheritance.\u201d<\/p>\n\n\n\n
Nu\u00f1ez further noted, \u201cI think our tool really allows us to begin to study the mechanism of heritability, especially epigenetic heritability, which is a huge question in the biomedical sciences.\u201d<\/p>\n","protected":false},"excerpt":{"rendered":"
Scientists led by a team at the Whitehead Institute have developed a new gene editing technology, called CRISPRoff, which can be used to control gene expression with high specificity, while leaving the sequence of the DNA unchanged. Utilizing a programmable, epigenetic memory writer protein to silence gene expression, CRISPRoff is a highly specific method that […]<\/p>\n","protected":false},"author":1,"featured_media":1092,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"categories":[22],"tags":[],"class_list":["post-1091","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-news_updates"],"yoast_head":"\n