Gene editing has been at the crux of improving life across all living organisms on Earth. When we think about science fiction novels and movies, we often fantasize about challenging the rules of nature itself, improving upon the millions of years of evolution that gave rise to the DNA and RNA sequences shaping the ecosystem we call planet Earth. Through our innate curiosity, humans realized that manipulating genes could correct flaws that still affect our survival today. The discovery of CRISPR systems gave us a tool capable of accomplishing what once seemed like science fiction: a repurposed bacterial system now used to potentially cure genetic diseases, build diagnostic tools, and engineer crops. It was like handing humanity a superpower, one capable of helping not just people, but the entire ecosystem. That possibility is what drew the Jain Lab to CRISPR engineering in the first place.
As a lab, we're constantly exploring different Cas proteins. Normally, CRISPR-Cas systems use RNA guides to find and cut DNA or RNA targets, depending on the protein, a rule that had gone unchallenged through thousands of published studies. In 2023, while working with Cas12i1, a relatively unexplored protein, we found something no one had shown before: it could use a DNA guide, instead of RNA, to target RNA. It sounds like a small flip, but it broke an assumption the field had held since CRISPR was first discovered.
We soon found a second protein, AsCas12a, could do the same thing. At first, we thought this new form of RNA targeting might make a useful diagnostic tool. But we suspected it could be something bigger if we could show it worked inside living cells, not just in a test tube. Many technologies that succeed in vitro fail to translate into cells, so we approached this cautiously. To our excitement, our early experiments worked. After building a reliable reporter assay, we moved on to testing RNA targeting of natural genes inside cells, which also succeeded. We had accomplished something we hoped for but the puzzle was still missing several pieces.
Whenever you discover a biological system that challenges conventional thinking, you have to characterize it as thoroughly as possible. Over the next stretch of the project, we learned and implemented techniques we never used before, Ribo-Seq, CLIP-Seq, PLA, BLI, and others. This to not only to gave a thorough explanation to the readers, but to convince ourselves that what we were seeing was real. It's a strange thing, fully accepting that you've done something you once thought might be impossible. That work let us comprehensively characterize the system: how it behaves in vitro through kinetic and binding analyses, and how it behaves inside cells. It also explained something puzzling, why a protein that targets RNA without directly cleaving it could still trigger endogenous degradation pathways that lower RNA levels.
With the mechanism mostly understood, one piece of the puzzle remained: an application that would take the project beyond a novel curiosity. We already knew the system itself was significant, the first DNA-guided CRISPR system to function inside cells while being less toxic to the transcriptome than existing systems. But we wanted more.
What we landed on was the first dual DNA-RNA targeting system driven by a single protein. Using one Cas protein, we could edit a DNA sequence and simultaneously lower the RNA levels of a target gene, with low off-target effects. It's hard to overstate what that meant in the moment: a single tool that could act on the genome and the transcriptome at once, something the field hadn't seen before. It opened the door to editing a disease-causing gene and dialing down its harmful RNA output in a single step. The kind of application that turns a mechanistic finding into a platform.
That left the final piece of the puzzle: what did this system actually look like? A cryo-EM structure was essential to visualize how it worked at the molecular level. We found a new functionality and demonstrated what it could do, but we still needed the structural confirmation to complete the picture. Thanks to our collaborator David Taylor and his team, we were finally able to see it and the puzzle, three years after it began, was whole.
Looking back, this project was driven by curiosity as much as ambition. What began as a simple question about whether CRISPR systems could function with DNA guides instead of RNA evolved into a discovery that challenged one of the field's foundational assumptions. From 2023 to 2026, piece by piece, we were reminded that the biggest discoveries aren't always about creating something entirely new. Sometimes they're about realizing that nature was more flexible all along, and that CRISPR, once science fiction itself, still has unexplored corners waiting for someone curious enough to look.
We hope this work expands the CRISPR toolbox, and encourages others to keep exploring the unknown spaces of biology where the next paradigm shift may already be waiting.