When the potential of CRISPR was first identified, its abilities seemed limitless as if it could be used to accomplish anything. But, in reality, those early days saw a very limited CRISPR that could only be inserted into a few kinds of cells and could only make a few narrow types of change accurately. The scientific community managed to improve on that imperfection over time and find new flashes of brilliance on how to better focus the original design. Since the purpose of CRISPR as a bacterial defense mechanism is far removed from how we are utilizing it today. Each subsequent advancement expanded the capabilities of CRISPR into an even more massive library of variant tools for specific types of genetic changes, from DNA to RNA to epigenetic manipulation.
However, there was always one major ongoing limitation: the scope of how CRISPR could be applied to a living organism. And, sure, there were technologies like gene drives that are able to cause a genetic change across a population and through generations, but that wasn’t the primary hope for CRISPR when we first found it. We want to change living organisms in the here and now, wholly alter their genome. Using CRISPR as a genetic medical tool on a grand scale requires that we are able to get the change to happen across a wide area of cells and alter all of them if we want the change to stick. Which has in itself been a major sticking point.
Retrons: Bringing Retro Back
Certain isolated conditions, such as harmful mutations in the eyes or missing a specific protein produced for proper muscle growth are all specific and narrow enough that we’ve made massive progress in curing them with CRISPR. The more systemic diseases though are a trickier question to answer with the tool as it is now. And that is something that researchers at the Gladstone Institute decided they would take a step toward solving by going right back into the bacterial toolbox that CRISPR had originally come out of.
What they pulled back from that box is another defense system of the microbial world referred to as retrons. These pieces of DNA engage in reverse transcription in order to counteract the damage done by bacteriophages that invade bacterial cells. In order for a bacteriophage to be successful in taking over a host cell, they must first undermine the workings of other defense systems that actively break down linear strands of DNA, such as what the bacteriophage genomes are made of. So the phages need to bind to these defensive complexes and inactivate them to continue their task of invasion. And it is at this point that the retrons take the stage.
Once the anti-phage complexes are turned off, the retrons begin their reverse transcription to start the production of DNA sequences that make toxic proteins. This toxicity, in turn, causes a bacterial community-saving process called abortive infection that kills the bacterial cell itself. The loss of one for the health and safety of the many. And the retrons do their reverse transcription process at a high efficiency, creating numerous copies of the needed DNA sequence in order to ensure the toxic products are thereafter transcribed and translated. This characteristic is the key that the researchers desired to exploit.
Making Retro New Again
Past research had noticed the utility of retrons for gene editing and had done some testing in both bacterial and eukaryotic cells. But the Gladstone Institute scientists wanted to go a bit further and alter the retrons on a larger scale than previously done in order to maximize the reverse transcription production of desired DNA. This would ensure more cells would have an abundant amount of the rtDNA to allow for a co-packaged CRISPR complex to do the alterations it was needed for. And they wanted to show that this could be successful in a variety of cell types, not just bacteria, but also eukaryotic yeast and mouse tissue cells.
The designer retrons they coded the genetic sequences for were made to have a universal architecture that would work across all the cell types they would be testing and could be generalized to use in any other tissue that future researchers desired. A key factor they found is that by extending the nucleotide length of the retrons in a specific manner, they created a more abundant amount of reverse transcribed DNA. They then confirmed that doing this increased the amount of available template sequences for the CRISPR complex to use and this, in turn, increased the efficiency and amount of cells edited.
A New Path Forward
While further optimization is required to make the perfect retron-CRISPR package for use in human cells, they have nonetheless proven that their new creation works in human cells and presumably all other species as well. The manipulation of retron lengths also allows for control over precise editing rate in a group of cells, allowing for case specific tweaks for the amount of cells that need to be targeted for different conditions. And retrons can be combined with all other gene editing tools beyond just a CRISPR-Cas9 complex.
So we now not only have a much greater versatility in gene editing technologies, but also a consistent control in treatment that can be tailored for particular medical conditions and the results kept repeatable for multiple patients. This was not at all the case with prior CRISPR tools that could have a different amount of treatment efficiency between people treated. Hopefully retrons will prove to be the missing piece to the medical cures we’ve been dreaming of using CRISPR gene editing for.
Photo CCs: CRISPR Cas9 (41124064215) from Wikimedia Commons