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Move Over CRISPR – RLR Shows Promise as a Better Gene Editing Technique

A colorful DNA strand

CRISP-Cas9 has made big news in the last few years. This relatively new gene editing technique enables scientists to selectively alter cell DNA. In the process, foods are being made more nutritious and healthier. Treatments for some genetic diseases are being developed as are warning markers for early detection. And an array of precision medicine considerations is being explored through enhanced DNA sequencing. But CRISPR has some features that make it less than ideal in some instances. This is why some researchers are exploring new gene editing techniques, and RLR looks to be a promising one.

RLR stands for Retron Library Recombineering. This gene editing technique leverages bacteria’s natural cellular machinery to evaluate millions of different DNA sequences. As a result, the ability for scientists to accelerate potential discoveries about different DNA codes is significantly improved. Better yet, RLR provides an inherent way to track each of these mutant DNA sequences. This allows scientists to better identify which ones might be most effective for precision medicine and other applications. For these reasons, many researchers are excited about the opportunities that RLR offers.

“RLR enabled us to do something that’s impossible to do with CRISPR: we randomly chopped up a bacterial genome, turned those genetic fragments into single-stranded DNA in situ, and used them to screen millions of sequences simultaneously.” – Max Schubert, PhD., Wyss Institute for Biologically Inspired Engineering

How RLR Gene Editing Works

Retrons are unique little packets of genetic material and enzymes within bacterial cells. Each consists of a single strand of RNA, single strand of DNA, and the enzyme reverse transcriptase. The enzyme interacts with the RNA to make changes to the single stranded DNA naturally. Interestingly enough, however, is that scientists are not exactly sure what this process does for a bacterial cell. Retrons may play a role in a bacteria’s ability to cause disease. Alternatively, they may also be important in how the cell uses energy or adapts to its environment.

While the actual purpose of retrons in bacterial cells is undefined, their potential in precision medicine is evident. Scientists can use retrons to create millions of different single-stranded DNA segments and then test each one. Gene editing using retrons provides an incredible number of DNA variants, called DNA mutants. And each one may have different effects that might be useful in precision medicine, enhanced nutritional foods, or other areas. (Learn more about gene-edited foods in this Bold story) Also, each retron-created DNA mutant sequence serves as its own tracking material. Like bar codes on packages, the unique DNA sequence lets scientists know exactly which one offered the best result.

“We figured that retrons should give us the ability to produce ssDNA within the cells we want to edit rather than trying to force them into the cell from the outside, and without damaging the native DNA, which were both very compelling qualities.” – Daniel Goodman, PhD., Jane Coffin Childs Postdoctoral Fellow at UCSF

How RLR Might Be Better than CRISPR

Without question, CRISPR-Cas9 techniques have been tremendous in revolutionizing gene editing approaches. (For deeper dive into CRISPR therapeutics, check out this Bold story.) However, these tools are not without some limitations. In essence, scientists use CRISPR to cut select DNA sequences from cells and replace them with others. In the process, through trial and error, they identify the best sequence for the desired effect. This has been used to create decaffeinated coffee beans, fruits with higher nutritional value, and treatment of muscular dystrophy. The precision medicine uses alone of CRISPR are believed to be enormous.

Someone editing a gene with an ink pen
Gene editing may be dominated by the CRISPR technique, but RLR is showing promise, too.

Unfortunately, CRISPR can be difficult to acquire in large quantities, which limits the ability to use it in research settings. Also, the Cas9 component, which cuts the DNA are precise locations is not always foolproof. It can also damage or cut DNA sequences outside the intended regions. CRISPR also requires introduction of new DNA sequences into the cell rather than from within. In each of these areas, RLR looks to be better. It permits intracellular DNA changes, allows millions of experiments to be performed at once, and is highly precise. As a gene editing tool, RLR holds greater promise in identifying new precision medicine treatments.

“Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other.” – George Church, PhD., Faculty, Wyss Institute for Biologically Inspired Engineering

Recent Research Involving RLR

While the gene editing technique involving RLR has been known for a few years, recent research has revealed its potential. In studies conducted at the Wyss Institute at Harvard, scientists have been examining the feasibility of RLR in E. Coli bacteria. Their protocol showed that E. Coli took up 90 percent of the retrons containing mutant DNA overall. As a result, the ability to detect mutations that caused antibiotic resistance was markedly accelerated. From the standpoint of precision medicine, this has great promise in identifying disease causes and therapies.

The downside to date is that RLR gene editing has only been demonstrated in bacterial cells. The same process has not yet been tested effectively using mammalian cells where precision medicine benefits may lie. Thus, while RLR may eventually prove to be much better than CRISPR, the latter remains the preferred approach currently. CRISPR has been shown feasible in plant and mammalian cells despite its limitations. In time, hopefully researchers can show that RLR is similarly an option in these settings.

RLR and the Future of Precision Medicine

Both CRISPR and RLR share one important thing in common beyond the fact that they can be used for gene editing. Both reflect inherent cellular processes that bacteria use to enhance their survival. CRISPR is believed to help bacteria fight off viruses by cleaving genetic material from threatening viruses for future viral identification. RLR is thought to provide bacteria with some advantages in adapting to its environment. But in terms of precision medicine, both give us opportunities to find genetic combinations that provide insights and cures. If RLR is faster and more thorough, then there’s little question it will be the prefer gene editing approach. This may not only be true for precision medicine but for a variety of applications.


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