Bruce Sullenger published a succinct review of the history and recent progress of RNA-guided endonuclease (RGEN) editing of RNA and DNA on Cell. Here are my learning notes.
- Three types of RGENs
- From RNA to DNA, from bacterial to mammalian
- Clinical studies with CRISPR-Cas9
Sullenger was one of the people that discovered RGEN and accompanied its development. The article introduces both history and the current status of investigating the potential of RGEN as therapeutics. The part of its application in the clinic is especially timely and interesting for me.
Three types of RGENs
There are different variants of RGEN editing, named Group I RGEN (RNA editing), Group II RGEN (DNA editing with exon binding sequences, or EBS), and CRISPR sgRNA-Cas RGEN (DNA editing with single guide RNAs, or sgRNAs). All of them undergo three common steps: binding, cleavage, and editing. It is enabled by the fact that an RNA molecule can guide an endonuclease to cleave another RNA at a specific nucleotide position.
The endonuclease can be used to cleave RNA molecules, for instance those by pathogens. The two major challenges associated with this approach are (1) the RNA cleavage products generated inside cells by endonucleases are not stable, making it challenging to study and optimize the approach, and (2) RGENs have to cleave the vast majority of a target RNA to produce the desired phenotype.
Then the idea came to let the RGEN not only cleave target RNAs, but also repair the broken phosphodiester backbone of a target RNA, editing the sequence in this process. This is the time when the three steps described above, binding, cleavage, and editing become stable.
From RNA to DNA, from bacterial to mammalian
The next questions were whether RGENs can edit RNAs in mammalian, especially human cells. And whether RGENs can edit DNA under physiological conditions. As we know, we can achieve both under certain constraints.
RNA targeting is likely more meaningful when transient editing of a nucleic acid is meaningful, since a long-lasting editing in DNA would be otherwise desired more. For instance, it can be used to reprogramm human genes into exogenous kinase to convert prodrugs into active drugs. In another example, the group I RGEN was delivered with adenoviral gene transfer, and the RGEN contains a liver-specific microRNA repressor sequence and a tissue-specific promoter to localize RNA editing to cancer cells. Most of such activities are applied in cancer now.
Group II RGENs can edit DNA in bacterial and in cytoplasm of mammalian cells but not in nucleus, likely due to the low concentration of Magnesium ions (Mg2+) there. Then the game changer, the CRISPR-Cas9 complex, came. The CRIPSR-Cas9 complex can be converted into a RGEN that can be programmed to cleave a specific DNA sequence through an RNA guide, known as sgRNA.
Clinical studies with CRISPR-Cas9
Two clinical studies with CRISPR-Cas9 were discussed. In one study, it was use to target the HIV-1 co-receptor CCR5. Despite of apparent good tolerability fater 19 months, only around 5% of the cells contained disrupted CCR5 genes. In another study with T cell receptor (TCR)-engineered T cells. The modified cells persisted for 9 months. Mild toxicity and chromosomal translocation was observed. The author concludes that “no major adverse events have been observed”, though he also proposes that long-term efficacy and safety profiles need to be evaluated in larger and longer trials, with special focus on unintended genome editing events, such as DNA translocations.
In conclusion, I think the article provides a compact history of discoverying RGENs and their applications as therapeutic agents. It is a timely and complementary read to the recent review The promise and challenge of therapeutic genome editing by Jennifer Doudna.