RNA Editing Possible with CRISPR-Cas13

Scientists extend the capabilities of the CRISPR-Cas system to include precise manipulations of RNA sequences in human cells.

By | October 25, 2017

WIKIMEDIA, NICOLLE RAGER, NATIONAL SCIENCE FOUNDATIONFusing an RNA-editing enzyme to an RNA-targeting Cas protein has enabled researchers to edit specific nucleotides within RNA molecules in human cells. The approach, called RNA Editing for Programmable A-to-I replacement (REPAIR), is described today (October 25) in Science, and has the potential to serve not only as a research tool, but as a temporary correctional therapy for disease-causing mutations, the researchers propose.

“This work is an impressive study from a highly productive research group that suggests the possibility of editing RNA transcripts to alter their coding potential in a programmable manner,” David Liu, a chemical biologist at Harvard University who was not involved in the project, writes in an email to The Scientist. “For applications that are best addressed through a transient change in a target RNA's sequence, this approach has strong potential,” he adds. Liu himself has a report out today in Nature describing specific nucleotide editing of DNA by a similar method.

See “Base Editing Now Able to Convert Adenine-Thymine to Guanine-Cytosine

The CRISPR-Cas9 system—a bacterial antiviral immune mechanism first discovered in Streptococcus thermophilus—is now widely used as a DNA editing technique, wherein the DNA nuclease Cas9 is directed to cut any DNA sequence of choice. By searching for similar immune systems in other microorganisms, researchers went on to discover a Cas9-related enzyme called Cas13 (originally termed C2c2), which, in 2016, the Broad Institute’s Feng Zhang and colleagues revealed targeted RNA not DNA.

Further developments with Cas13 led to a Nature paper earlier this month in which Zhang’s team showed that one family member, Cas13a, could be used in mammalian cells to knockdown particular messenger RNAs with similar efficiency to RNA interference. The team also engineered a catalytically inactive version of Cas13a that, when fused to a fluorescent protein, could be used to track RNAs of interest.

See “CRISPR System Targets RNA in Mammalian Cells

In the new study, Zhang ‘s team asked, “What else can we do with a catalytically dead Cas13 enzyme?” says Omar Abudayyeh, a student in Zhang’s lab and coauthor of the study. One idea was to use the protein’s RNA-targeting capacity to recruit an RNA-editing enzyme that would allow the researchers to make defined nucleotide alterations at specific sites.

RNA editing is a form of transcript processing involving, among other things, enzymatic regulation of nucleotide substitutions. Enzymes called adenosine deaminases acting on RNA (ADAR), for instance, convert adenosine into inosine—a nucleotide that is functionally equivalent to guanosine. Zhang’s team has now created a fusion protein between the catalytic domain of ADAR and a catalytically inert Cas13b—another Cas13 family member—with the ability to target any RNA of interest in human cells. Indeed, they used it to revert two disease-associated G-to-A mutations—one that causes a form of diabetes, and another that causes Fanconi anemia—in two separate messenger RNAs.

Therapeutically speaking, for certain genetic diseases, editing the genome to permanently fix a mutation may be more desirable. But RNA editing might be preferable “in situations that require only short-term changes in gene expression,” suggests RNA biologist Mitchell O’Connell of the University of Rochester in New York who did not participate in the research. As examples, he lists “organ transplants, where a short phase of rejection prevention may be necessary, temporary reductions in inflammation, and certain acute diseases.”

The immediate power of the REPAIR system, O’Connell says, is as a research tool. Introducing specific sequence changes into RNA molecules could allow researchers to answer questions about alternative splicing mechanisms, translation, and even editing, he says. “There’s a lot of scope.”

The tool itself could be further developed, adds computational biologist Eugene Koonin of the National Center for Biotechnology Information who also was not involved in the study. “This paper is not the end of the road,” he says. It’s possible that Cas13b could be fused to a variety of editing enzymes that would allow a range of different sequence changes. The possibilities are numerous, Koonin says, and “the best is still to come.”

D.B.T. Cox et al., “RNA editing with CRISPR-Cas13,” Science, doi:10.1126/science.aaq0180, 2017.

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Avatar of: dumbdumb

dumbdumb

Posts: 94

October 25, 2017

Hugely overhyped findings


- If someone has a disease that can be DNA/RNA edited, I am pretty sure s/he would rather have it corrected permanently.


- The transitory correction can be achieved by "old" school RNA interference (one RNA to block the defective one, one with the functional sequence). The problem is in getting it in to the right cells. Which is exactly the same for the CRISPR technology


 

Avatar of: James V. Kohl

James V. Kohl

Posts: 481

October 26, 2017

Introducing specific sequence changes into RNA molecules could allow researchers to answer questions about alternative splicing mechanisms, translation, and even editing, he says. “There’s a lot of scope.”

The entirety of that scope was addressed in the context of energy-dependent RNA-mediated cell type differentiation in our section on molecular epigenetics from this 1996 Hormones and Behavior review. From Fertilization to Adult Sexual Behavior

Small intranuclear proteins also participate in generating alternative splicing techniques of pre-mRNA and, by this mechanism, contribute to sexual differentiation in at least two species, Drosophila melanogaster and Caenorhabditis elegans (Adler and Hajduk, 1994; de Bono, Zarkower, and Hodgkin, 1995; Ge, Zuo, and Manley, 1991; Green, 1991; Parkhurst and Meneely, 1994; Wilkins, 1995; Wolfner, 1988). That similar proteins perform functions in humans suggests the possibility that some human sex differences may arise from alternative splicings of otherwise identical genes.

All biophysically constrained RNA-mediated energy-dependent cell type differentitaion has since been linked from the pheromone-controlled physiology of reproduction to supercoiled DNA via the fixation of amino acid substitutions and chromosomal rearrangements.

The facts about the amino acid substitutions in the context of transgenerational epigenetic inheritance link electrons to ecosystems via the cryo-EM technology that won the 2017 Nobel Prize in Chemistry.


 

Avatar of: James V. Kohl

James V. Kohl

Posts: 481

October 29, 2017

The possibilities are numerous, Koonin says, and “the best is still to come.”

For people like Eugene Koonin, the worst is yet to come. By playing the cell biology game "Cytosis," anyone over age 10 will learn that energy is required to biophysically constrain the virus-driven degradation of messenger RNA that links mutations to all pathology in species from microbes to humans.

Previously, Koonin reported that The entire evolution of the microbial world and the virus world, and the interaction between microbes and viruses and other life forms have been left out of the Modern Synthesis...

That fact suggests he is trapped with untested theories like those that Feynman claimed were examples of human idiocy.  See: Food energy


 

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