All-RNA Gene Writing Method Paves Way for Precise Gene Editing in Human Cells

A research team from the Chinese Academy of Sciences' Institute of Zoology has created a novel gene-writing technology based on retrotransposons, as reported in a recent study published in Cell. This accomplishment permits targeted gene integration in human cells through all-RNA-mediated means.

One of the main challenges in genome engineering is the efficient and accurate integration of gene-sized DNA. For gene integration, current technologies mostly use DNA templates as donors. However, in real-world biomedical applications, DNA donors encounter many obstacles, including high immunogenicity, challenges with in vivo delivery, and the possibility of random integration into the genome.

However, RNA donors are less immunogenic than exogenous DNA donors. RNA donors can be delivered efficiently through non-viral vectors and are quickly broken down in cells without the possibility of random integration, thereby resolving many of the issues with DNA donors. However, as of right now, very few technologies are able to integrate targeted gene-sized DNA into human cells using RNA donors.

R2 retrotransposons are mobile elements that specifically integrate into the host 28S rDNA genomic site through the use of RNA intermediates. This site is found in the human genome in 219 copies, and it is a "safe harbor" that can be used for gene integration because it is not close to genes that code for proteins. Its potential for integrating large-fragment genes into human cells has not yet been thoroughly investigated, despite its discovery in the 1980s.

In this study, the R2Tg system derived from the avian genome was found to be active, albeit inefficiently, in human cells by means of systematic analysis and screening. Through a variety of engineering techniques, the researchers developed en-R2Tg, an enhanced variant of R2Tg.

The en-R2Tg gene-writing tool can achieve 25% gene integration efficiency in human liver cells and over 60% site-specific gene integration efficiency in mouse embryos when delivered via lipid nanoparticle (LNP), a non-viral vector used in clinical settings.

Furthermore, at the 28S rDNA safe harbor site, the en-R2Tg system demonstrates high gene integration specificity. This reduces the possibility of mutagenesis brought on by the random integration of genes produced by technologies like retroviruses.

In conclusion, this work creates an accurate, efficient, and all-RNA mediated gene-writing technology based on R2 retrotransposons that are found in nature.

This technology opens a door for the development of novel gene therapeutics. When it comes to a disease-related gene, there can be many different mutations that cause the same disease. Our technology offers a more general approach in which we can integrate a normal gene directly into the genome to restore function, regardless of the type of mutation.”

LI Wei, Study Corresponding Author, Institute of Zoology, Chinese Academy of Sciences

“We may even be able to use LNP to deliver our gene-writing tool and create CAR-T cells directly in our bodies to treat cancer. This could make the whole process as easy as getting a vaccination. We’re excited about the potential for further development and application of this new technology in the future,” said Li.