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The new CRISPR platform expands the capabilities of RNA editing

The new CRISPR platform expands the capabilities of RNA editing

The CRISPR Cas13 family enzyme at work. Cas13 (pink) is at the heart of the RESCUE platform, where she uses a special guide (red) to target RNAs in the (blue) cell. Credit: Stephen Dixon

CRISPR tools have revolutionized our ability to target genetic mutations related to diseases. CRISPR technology includes a growing family of tools that can manipulate genes and their expression, including targeting DNA with Cas9 and Cas12 enzymes and targeting RNA with the enzyme Cas13. This collection offers different strategies to fight against mutations. Targeting disease-related mutations in RNA, which has a relatively short lifespan, would prevent permanent genome changes. In addition, it is difficult to edit certain types of cells, such as neurons, using CRISPR / Cas9 mediated editing, and new strategies are needed to treat the devastating diseases that affect the brain.

The researcher at the McGovern Institute and the senior member of the MIT and Harvard Main Institute, Feng Zhang, and his team have come up with a strategy of this type, called RESCUE (Edition of ARN for a specific exchange C to U), described in the journal Science.

Zhang and his team, whose first co-authors, Omar Abudayyeh and Jonathan Gootenberg (both now members of McGovern Society), have used a deactivated Cas13 to guide rescue on cytosine bases targeted to transcripts of the disease. RNA, and used a new scalable and programmable enzyme to convert. undesirable cytosine in uridine, resulting in a modification of the instructions of the RNA. RESCUE relies on REPAIR, a technology developed by Zhang's team to turn the basics of adenine into inosine into RNA.

RESCUE significantly expands the landscape that CRISPR tools can target to include for the first time modifiable positions in proteins, such as phosphorylation sites. These sites act as on / off switches of protein activity and are found in particular in signaling molecules and cancer-related pathways.

"To address the diversity of genetic changes that cause disease, we need a range of specific technologies.While developing this new enzyme and combining it with the programmability and precision of CRISPR, we have been able to fill a gap critical in the toolbox. "says Zhang, neuroscience professor James and Patricia Poitras at MIT. Zhang also has positions in MIT's Brain and Cognitive Science and Biological Engineering Departments.

Extend the scope of the RNA edition to new targets

The previously developed REPAIR platform used the CRISPR / Cas13 targeting RNA to direct the active domain of an RNA editor, ADAR2, to specific RNA transcripts where it could convert adenine from the nucleotide base to inosine, or the letters from A to I. If you existed with alternative activities, Zhang and his colleagues took the fusion REPAIR and developed it in the laboratory until it can change from cytosine to uridine, or from C to U.

RESCUE can be guided to any RNA of your choice, and then perform a C-to-U edition via the ADAR2 Advanced Component of the platform. The team introduced the new platform into human cells, showing that they could target natural RNAs in the cell as well as 24 clinically relevant mutations in synthetic RNAs. They then optimized RESCUE to reduce out-of-target changes, while minimizing target changes.

New targets in sight

The extended targeting by RESCUE means that sites regulating the activity and function of many proteins through post-translational modifications, such as phosphorylation, glycosylation and methylation, can now be targeted for the first time. edition.

One of the main advantages of editing RNA is its reversibility, unlike the changes made at the DNA level, which are permanent. Thus, RESCUE could be deployed transiently in situations where a modification may be desirable temporarily, but not permanently. To demonstrate this, the team has shown that in human cells, RESCUE can target specific sites of RNA encoding β-catenin, which are known to be phosphorylated on the product. protein, resulting in a temporary increase in β-catenin activation and cell growth. If such a change were made permanently, it could predispose cells to uncontrolled growth and cancer, but using RESCUE, transient cell growth could potentially stimulate wound healing in response to acute lesions.

The researchers also targeted a variant of the pathogenic gene, APOE4. The APOE4 allele has consistently emerged as a genetic risk factor for the development of late-onset Alzheimer's disease. The APOE4 isoform differs from APOE2, which is not a risk factor, by only two differences (C in APOE4 and U in APOE2). Zhang and his colleagues introduced the risk-associated APOE4 RNA into the cells and showed that RESCUE can convert its C signatures into an APOE2 sequence, converting a risk into a risk-free variant.

To facilitate the extra work that will push RESCUE to the clinic and allow researchers to use RESCUE as a tool to better understand disease-causing mutations, the Zhang laboratory plans to share the RESCUE system in general, as it does. did with the CRISPR tools developed previously. The technology will be freely available for academic research through the Addgene Nonprofit Plasmid Deposit. Additional information is available on the Zhang Lab website.

CRISPR engineering researchers to edit simple letters of RNA in human cells

More information:
"A cytosine deaminase for programmable single-base RNA editing" Science (2019). science.sciencemag.org/lookup/… 1126 / science.aax7063

Provided by
Massachusetts Institute of Technology

The new CRISPR platform expands its RNA editing capabilities (11 July 2019)
recovered on July 12, 2019
from https://phys.org/news/2019-07-crispr-platform-rna-capabilities.html

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