"Nature Biotechnology" RNA editing has reached a new level!

The treatment of genetic diseases has always been a difficult problem to be solved

Fortunately, with the development of human genomics, more and more human genetic disease genes have been discovered one after another

It has become a promising treatment to fundamentally cure human genetic diseases by altering the genetic sequence of patients with genetic diseases

More recently, the development of various programmable, site-specific tools for processing genetic information has created opportunities to correct disease-causing genetic mutations, such as the DNA base editor developed by David Liu et al

However, it is worth noting that DNA editing can lead to inherited and permanent off-target mutations, which greatly limits the application of DNA base editors in humans

Recently, the team of Professor Thorsten Stafforst of Tübingen University in Germany published a research paper entitled "CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo" in the journal Nature Biotechnology

This study develops a set of guide RNAs, gRNA clusters, that bind target mRNAs in a multivalent manner without the need for exogenous proteins, utilize endogenous ADAR enzymes to enable high-precision and high-efficiency editing, and enable editing that cannot be achieved by gRNA design targeted editing of previous sequences

In cell experiments, the editing efficiency of CLUSTER gRNAs is as high as 45%, and in mouse experiments, the editing efficiency of CLUSTER gRNAs is also as high as 10%

RNA base editing is a promising alternative to genome editing

These RNA editing tools require the ectopic expression of an engineered editing enzyme, potentially resulting in substantial off-target editing

Indeed, ectopic expression of the highly active A-to-I editing enzyme leads to lethal consequences in a fragile mouse model of the disease

Based on this, more and more studies use endogenous RNA-specific adenosine deaminase (ADAR) to solve the problem of ectopic expression of engineered editing enzymes, but there are still problems such as sequence limitations, low editing efficiency, and off-target editing

As early as October 2016, Professor Stafforst's team published a related study on the use of endogenous ADAR to edit RNA in nucleic acid research

The title of the article is: Harnessing human ADAR2 for RNA repair – Recoding a PINK1 mutation rescues mitophagy

The team designed a special gRNA that contains complement that binds to the target sequence, as well as RNA structural motifs that recruit ADARs

The design was particularly successful when these gRNAs were used in the form of chemically modified oligonucleotides, yielding 20-30% edits to related transcripts such as STAT1

Notably, the researchers found that the same oligonucleotide sequences were much less effective when expressed in plasmids
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One possible explanation is that chemically modified oligonucleotides have stronger binding affinity
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Thus, plasmid-borne gRNAs successfully edited endogenous targets via endogenous ADARs when the specificity domain was extended beyond 100 nt
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Thorsten stafford refers to these gRNAs as LEAPER gRNAs, however, the LEAPER approach shows substantial off-target editing because longer gRNAs bind more readily to off-target mRNAs
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Therefore, the research team improved it and described the design principles of new CLUSTER gRNAs - including a specificity domain that binds to target mRNA and a set of 7-20nt recruiting sequences (RS), as well as ADAR recruiting sequences (RS) RNA structural motifs
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This CLUSTER design enables genetically encoded gRNAs with a high degree of sequence flexibility to bind to their target mRNAs in a multivalent manner, enabling high-precision, high-efficiency editing, and the ability to target sequences not achievable using previous gRNA designs
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CLUSTER gRNAs can be delivered by genetically encoded and viral vectors for efficient editing in a variety of cell lines
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In cell culture, CLUSTER gRNAs are capable of targeted editing of endogenous transcripts with efficiencies as high as 45%
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In vivo, infusion of CLUSTER gRNAs into mouse liver via hydrodynamic tail vein injection (HTVi) resulted in up to 10% of edited reporter gene construction
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In conclusion, this study developed a CLUSTER gRNAs that recruit endogenous ADAR enzymes for RNA editing with higher editing efficiency and fewer off-target effects
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The CLUSTER approach opens up new avenues for drug development in the field of RNA base editing!

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