The Yichengji research group in the School of Life Sciences has developed a new RNA editing technology RESTART

On December 14, 2022, Professor Yi Chengji's research group from the School of Life Sciences of Peking University was present The journal Molecular Cell published online titled "CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.
" The first to report a novel RNA single-base editing technique
called RESTART (RNA Editing to Specific Transcripts for Pseudouridine-mediAted PTC-ReadThrough).
This technique utilizes engineered guide snoRNA to recruit endogenous pseudouridine synthase complexes within cells to achieve efficient and accurate uridine (U) to pseudouridine (Ψ) editing
at specific sites of RNA.
Pseudouridine modification is precisely introduced at the nonsense mutation site of mRNA, and the early stop codon is converted into ΨAA, ΨAG or ΨGA to achieve the read-through of the early stop codon and the full-length expression
of functional proteins.

Screenshot of the paper

A nonsense mutation is a single-base mutation
in a genetic sequence in which the codon encoding an amino acid mutates into a stop codon (TAA, TAG, TGA).
Nonsense mutations produce Premature termination codons (PTCs), resulting in early termination of translation, resulting in smaller, non-functional protein products
.
According to the Human Gene Mutation Database ( nonsense mutations account for more than 20% of disease-associated single-base mutations
.

There are a variety of potential techniques available to treat nonsense mutant diseases, but there are still limitations
.
For example: (1) CRISPR/Cas9-dependent DNA base editing technology can achieve precise base repair, but there are still safety issues
.
Bacterial-derived Cas proteins may trigger an immune response in humans; And once there is an off-target at the genomic level, it will be permanent
.
In addition, the large size of the editing element limits the delivery of the drug in vivo
.
(2) RNA base editing technology is carried out at the RNA level and does not permanently change the genome sequence, so it is safe
.
However, there are still safety risks
to the off-target effect of RNA editing tools.
Therefore, there is an urgent need to expand new RNA editing tools and develop more specific and safe RNA editors
.

PRINCIPLE OF RESTART TECHNOLOGY

Studies have shown that the RESTART technology has broad applicability
.
RESTART mediates efficient and precise editing
in many cell lines of different tissue origins, as well as in human primary cells, such as bronchial epithelial cells and skin fibroblasts.
In many application attempts for disease nonsense mutation repair and protein function recovery, the high efficiency of RESTART has been fully verified, reflecting the great potential
of this technology in disease treatment.
RESTART successfully restored the function
of the IDUA protein in α-L-iduronic enzyme-deficient cells derived from mice with Hurler syndrome.
This technology provides a completely new tool
for the treatment of nonsense-mutant diseases and for basic research on RNA pseudouridine modification.

Traditional RNA editing technology mainly achieves base editing through deamination reaction (such as A-to-I or C-to-U), and the off-target will introduce mutations on RNA, which is a safety risk
.
Unlike these techniques, pseudouridine modification does not change the base complementary pairing and does not affect the coding information of codons; The small amount of off-target generated by RESTART also does not affect RNA stability and protein translation
.
In addition, the RESTART system is derived from human-derived snoRNAs and modified enzymes, which theoretically avoid immunogenicity
.
Therefore, RESTART is a potential therapeutic technique
that is efficient and safe.

In conclusion, as a programmable CRISPR-independent RNA pseudouridine editing technology, RESTART technology expands the strategy of RNA editing, which can mediate translation read-through and protein function recovery by efficiently editing nonsense mutation sites on mRNA, and has good safety and shows good disease application prospects
。 In terms of delivery, RESTART is suitable for loading into vectors such as adeno-associated virus (AAV) for delivery; And guide snoRNA can be prepared by various methods such as in vitro transcription and in vitro synthesis, and can also be coupled
to small RNA delivery systems, such as_{GalNAc 3} in the future.
In addition, RESTART technology will also promote research in the field of pseudouridine modification, providing favorable tools
for basic research in this field and the treatment of nonsense mutational diseases.

Yi Chengqi is the corresponding author of the paper, postdoctoral fellow Song Jinghui (has exited), doctoral students Dong Liting, Sun Hanxiao, Luo Nan, and postdoctoral fellow Huang Qiang are co-first authors
.
This work is supported by the Ministry of Agriculture, the Key R&D Program of the Ministry of Science and Technology, the National Natural Science Foundation of China
, the Peking University-Tsinghua Joint Center for Life Sciences, and the State Key Laboratory of Protein and Plant Gene Research.
Peking University High Performance Computing Platform, School of Life Sciences Instrument Center and Phoenix Engineering and other platforms have provided important technical support
for this project.