The winters in Ithaca, NY, are long, snowy, and bitterly cold. The Cornell University students who endure them can be overheard discussing “Ithacation”—a mix of snow, hail, rain, and slush—as they trudge their way to class. But this past winter, a group of researchers on campus were focused on something besides the weather. Three labs, in two different departments, were racing to figure out the detailed mechanism underlying a CRISPR-associated transposition system.

Liz Kellogg, PhD, is the newest member at Cornell to focus on CRISPR, having joined the faculty as an assistant professor in 2019. A structural biologist by training, Kellogg came to Cornell from the lab of the renowned structural biologist David Baker, PhD, the Breakthrough Award winner who heads the Institute for Protein Design at the University of Washington.

Members of the Peters and Kellogg labs at Cornell University [Liz Kellogg]
Kellogg hadn’t previously met Joe Peters, PhD, professor in the department of microbiology at Cornell, before she moved to Ithaca. That’s not surprising—Peters is not a structural biologist but rather an expert on transposons—an area of research he says he has “loved for more than 30 years.” More recently, Peters has focused on one particular group of transposons—those that have CRISPR-Cas systems.

Together with Eugene Koonin, PhD, senior investigator in the evolutionary genomics research group at the National Center for Biotechnology Information (NCBI), Peters first discovered the link between CRISPR-Cas and transposons a few years ago. The first report of Tn7-like transposons that contain CRISPR-Cas systems was a joint 2017 paper in the Proceedings of the National Academy of Sciences. Since then, the jumping CRISPR systems have garnered intense interest and the pace of discovery has sped up.

Samuel Sternberg, PhD, Columbia University [MattWong]
In 2019, two papers—published within one week of each other—solidified Peters’ and Koonin’s hypothesis by elucidating the integration event of CRISPR associated transposons. The lab of Samuel Sternberg, PhD, assistant professor in the department of biochemistry and molecular biophysics at Columbia University, published one of the two papers. The second was the work of the lab of Feng Zhang, PhD, investigator at the McGovern Institute at MIT, and gene editing pioneer. Both papers showed that CRISPR associated transposons can insert large fragments of DNA without making double-strand breaks.

In their new paper, published this week in Science and titled, “Structural basis for target-site selection in RNA-guided DNA transposition systems,” the labs of Kellogg, Peters, with collaboration from their colleague and genome editing expert Ailong Ke, PhD, have probed deeper into the mechanisms of CRISPR-associated transposition.

Using a structural biology technique called cryo-electron microscopy (cryo-EM), the team teased apart the peculiarities of how the CRISPR-associated transposons choose their insertion site. The systems use a guide RNA but only insert in one orientation—the 5’->3’ direction—with very defined spacing from the guide RNA. These characteristics led the Cornell team to propose that there must be communication between the CRISPR domain and the transposon. In addition, the transposon targets essential genes. The transposon recognizes a target gene and inserts downstream of it, leaving only a couple of base pairs from the gene.

Cryo-EM produces maps that are used to produce models to help interpret the functional cycles of the system. Kellogg’s lab used this technique to characterize the transposition regulator, a protein named TnsC. Through this work, they not only elucidated the structure but were also able to characterize the functional cycle of TnsC, show how it loads onto DNA, how it finds its target site, and how it defines its precise spacing. In addition, they found that ATP hydrolysis is at the center of this process and is required for proper target site selection. They hypothesized that the polymerization of ATP-bound TnsC helical filaments could be involved in the transfer of information to the transposase.

Researchers have previously shown that TnsC is the target-site selector in the system. But Kellogg’s team was able to visualize it in a complex with the adaptor protein (TniQ) that interacts with the CRISPR effector domain. No one had visualized the interactions between these two proteins before and, Kellogg said they were not sure if it would form a stable complex. She said it was a “huge mystery” to her if they would be able to capture it, so not only was she surprised when they did so, but the assembly that was revealed was also unexpected.

The Science paper, noted Sternberg, presents “exciting structural advances” that clarify the structure and function of TnsC during RNA-guided DNA integration. One question left unexplored, said Sternberg, is how these data might shed light on the promiscuous integration specificity that has been observed for Type V CRISPR-transposons, as opposed to high-fidelity integration by Type I CRISPR-transposons. Could RNA-independent TnsC filament formation play a role?

Members of the Kellogg lab and Joe Peters, PhD [Liz Kellogg]
There will be many more answers (and likely more questions) to come. As for the CRISPR team at Cornell, they are trying to re-engineer these systems and make improvements; for example, to create a more faithful insertion tool by decreasing the system’s significant level of off-site targeting. Kellogg told GEN that there will be many new findings coming out soon from them, and others, and that the field promises some exciting years ahead.


Jumping on CRISPR-Tn systems

The interest in these CRISPR-transposase systems has grown widely over the past few years. One need look no further than the bioRxiv preprint server over the past week to find two related structural studies—posted by Leifu Chang’s group at Purdue University and Martin Jinek’s group at the University of Zurich, Switzerland—of TnsC and Cas12k.

Why the intense interest? Because these systems “tick all of the boxes,” said Peters, referring to using the genome editing tool therapeutically, including the ability to deliver large payloads to DNA without double-stranded breaks.

CRISPR-transposons, noted Sternberg, offer a fundamentally new approach to the targeted integration of large genetic payloads using RNA-guided targeting. Alongside base editors and prime editors, RNA-guided transposases “may increase the safety of certain genome editing experiments for human therapeutic applications.”

But to approach therapeutic applications, the system first needs to move into human cells—which hasn’t been reported yet. Kellogg said that this system, as it was first described in 2019, is not going to go into human cells as is but will require further optimization.

“We’re not there yet,” added Peters, casting assurances that many people have tried to put this system into human cells but, “it’s not going to be simple.” However, the advances in his team’s new report present a mechanism that brings them one step closer. “Now, we have a better idea of how it functions,” Peters noted.

The structural advances in this paper, said Sternberg, highlight intrinsic challenges in reconstituting these multi-component and multimeric systems in heterologous cell types. However, he added, “these are surmountable hurdles, not impenetrable barriers.”