April Pawluk

New from @arcinstitute: Megabase scale genome-editing in human cells. Bridge recombinases are a new class of genome editors. In this paper, they were used to invert up to 920,000 base pairs of DNA and also cut out 130,000 base pairs, "with no apparent distance dependency." Let me explain what these Bridge recombinases are, and why I think this is a big deal. There are dozens of CRISPR-based gene-editing tools that can be used to swap nucleotides in the genome, delete parts of genes, or replace sequences entirely. But they all rely on cell repair pathways to make their edits. If a researcher wants to permanently “shut down” a gene (using CRISPR-Cas9, for example), then Cas9 must go into the genome and make a cut at the position indicated by its guide RNA. The Cas9 doesn't then fix the DNA it has broken, however; the cell must do that a different way. There are two main ways to fix this damage. Non-homologous end joining quickly slaps the two broken strands together, often adding or deleting random bits of DNA in the process. For the second option, homology-directed repair, scientists can introduce a DNA "donor template" into cells alongside CRISPR-Cas9, and then the cell copies from this template to fix the break. But homology-directed repair only works reliably during specific phases of the cell cycle and happens less frequently than non-homologous end joining. Because CRISPR-based tools rely on these cellular repair pathways, edits are inherently unpredictable. Different cells will fix the damage in different ways, leading to different edits. (Prime editors get around these two major repair pathways, but rely on a third mechanism, called the mismatch repair pathway.) Here's the punchline: Bridge recombinases can make megabase-scale edits in human cells, and do so **without relying on cellular repair mechanisms** — unlike CRISPR-based tools — which could make them more predictable and versatile in the long run. This Bridge genome-editing tool is made from two parts: a protein (called the recombinase) that cuts and rejoins strands of DNA, and a RNA molecule (called the ‘Bridge’) that guides the recombinase to a specific location in a cell’s genome. (This tool was initially adapted from an existing transposase found in nature.) For this new paper, researchers used Bridge recombinase to precisely invert a 930,000 base-pair sequence in human cells, and also to chop out 130,000 bases in a single go. They also used Bridge recombinases to edit a gene linked to a disease called Friedreich’s ataxia. Whereas healthy people have several repeats of a sequence—GAA—in a gene called FXN, people with Friedreich’s ataxia have hundreds or thousands of the repeats. This causes the gene to make a defective protein that slowly causes nerve damage. In a cell culture model, the researchers used a Bridge recombinase to cut out more than 80 percent of the repeating sequences (and it worked about 40 percent of the time.) One immediate application for these Bridge recombinases is to make cancer cell lines in the laboratory. Many types of cancers are caused by large-scale genome rearrangements that can't easily be replicated with other genome-editing tools. Chronic myeloid leukemia, for example, happens when chunks of chromosomes 9 and 22 swap places. Using Bridge recombinases, researchers could recreate this rearrangement in healthy cells to study how it causes disease and how to reverse it. Ditto for Ewing’s sarcoma, a bone cancer caused by another type of chromosome fusion. There are other uses for Bridge recombinases, too. Scientists can use them to make just about any type of large-scale edits, which means that “genome design” is now an actual possibility. And so maybe the questions we started with—"How does this work? Why is it better?"—aren't even the right ones. For decades, biologists have mainly been observers; cataloging genes, making little mutations; mapping chromosomes; knocking stuff out and trying to put piecemeal observations together again. But now, for the first time, there is a tool good enough to rewrite large stretches of the human genome. So the questions worth asking have changed. Instead of wondering what these tools can do, we should start thinking about what we want to do.