NEW YORK – A new class of zinc finger DNA-binding domain (ZFD)-dependent recombinases outperform other genome editing tools in some respects through improved target specificity and reduced nonspecific enzyme activity.
The new recombinases, developed at the Technical University of Dresden, were described in a study recently published in Nature Biotechnology and have been licensed to TU Dresden spinout Seamless Therapeutics.
Recombinases catalyze DNA exchange interactions and have several potential advantages as genome editing tools compared to nucleases such as CRISPR-Cas9 and other editing enzymes, but engineering them to efficiently recombine specific DNA targets is more laborious.
Researchers at TU Dresden devised an approach to generating programmable ZFD-dependent recombinases that remain dormant unless linked to their target DNA sequence without inducing double-stranded breaks and without relying on the cellular DNA repair machinery or other co-factors.
"These recombinases are very, very, promising," said Frank Buchholz, head of translational research at the TU Dresden cancer center and senior author of the study, "because they do the whole recombination reaction all by themselves."
Buchholz added that their lack of reliance on any host proteins also means that they work in non-dividing cells.
"Recombinases also have the advantage that they use the Cre-lox [system] that has been used for decades to do conditional knockouts and has been safely used in many animals," Buchholz added.
To engineer these conditional recombinases, Buchholz's lab made fusion proteins consisting of a ZFD linked to a tyrosine site-specific recombinase (SSR) via a flexible linker. The ZFD then targets a specific DNA sequence and the SSR confers the enzymatic activity.
The team further optimized this method through extensive screening of potential constructs across a library of recombinase and ZFD target sites using nanopore long-read deep sequencing. The resulting enzymes displayed better DNA editing activity than the original parental recombinase.
A key discovery made during this process was that insertional ZFD fusions disrupt recombinase activity, while DNA editing activity can be recovered when recombinase target sites sit adjacent to the appropriate ZFD binding sites. This means that inserting a ZFD into the coding sequence of a recombinase effectively impairs recombinase activity at its target sites until the fused ZFD binds to its cognate motif flanking these sites.
"This is an important discovery, we believe," Buchholz said, "because this way we can now make conditional recombinases, where we can guide the recombinase to a certain place … then do the recombination reaction."
This conditional targeting strategy enabled Buchholz's team to exploit so-called "promiscuous recombinases," or those with relaxed target site specificity in their native form, as more precise genome editors.
One such recombinase identified in the course of directed evolution experiments, named RecFlex, appeared particularly promising, given its ability to recombine a range of sites.
Expressing such a recombinase in its native form, Buchholz said, would likely prove toxic to the cell because it could recombine potentially thousands of sites, "making a mess out of the genome."
The addition of the zinc finger, however, essentially "tames" the recombinase activity, making it's activation dependent on both the correct ZFD binding and target binding sites, which, Buchholz said, makes accidental recombinase activity "virtually impossible."
Luca Pinello, an assistant professor of pathology at Harvard University, whose research focuses on chromatin structure and dynamics, said that he was excited to see another method added to the genome manipulation toolkit.
"I'm particularly eager to see how this technology will evolve to meet challenges in the future" he said, "such as enhancing the scalability of designing custom recombinases for a variety of target sequences related to the ZFD design challenges and improving its efficiency across different cell types and organisms."
Pinello added that conducting a thorough off-target analysis to gather more data will be crucial before fully embracing Buchholz's approach in applied research or therapeutic applications.
Buchholz did, in fact, uncover some therapeutic potential for a RecFlex-based genome editor. Through an extensive genome-wide search, Buchholz's team pinpointed a clinically relevant RecFlex-like target site within the MECP2 locus on the human X chromosome. Duplication events at this site are directly implicated in the onset of the MECP2 duplication syndrome, a condition that occurs almost exclusively in males and is characterized by moderate to severe intellectual disability.
Most other genome editors are currently less costly and time-consuming than Buchholz's conditionally activated recombinase system, albeit with more off-target effects. Buchholz, however, thinks that his platform's greater target site specificity will make it competitive.
"It still takes more work than reprogramming your CRISPR enzyme," he said. "We use that a lot, but I think for the benefits [the recombinase system] has on the therapeutic level, the cost of making them are, in our view, negligible." His TU Dresden spinout Seamless Therapeutics, he noted, can now make a new recombinase within a month or so. "We've streamlined the process, automated a lot of the steps, and this is really not a bottleneck anymore," Buchholz said.
Seamless launched in March 2023 with $12.5 million in seed funding led by Wellington Partners and Forbion, and with non-dilutive financing from BMBF GO-Bio, a German government initiative founded to support innovative life sciences startups.
TU Dresden has exclusively licensed the intellectual property related to the conditionally activated recombinase system to Seamless. The company is using the technology to develop a pipeline of disease-modifying therapeutic candidates in multiple disease indications, but it is currently too early to disclose which disease areas the company is prioritizing.
The challenge of target specificity in genome editors is a topic of considerable academic and commercial interest, with numerous players testing a variety of diverse approaches in that space. Many of these revolve around prime editing. Prime editing works by combining the DNA-scanning and sequence-identification capabilities of CRISPR-Cas9 with a reverse transcriptase enzyme, which uses an RNA template to synthesize and insert a new single-strand DNA sequence.
The Broad Institute, for instance, recently developed a prime editing method that enables more precise DNA sequence replacement and excision, also without the need for double-strand DNA breaks.
Chinese life sciences company Qi Biodesign is also exploring prime editing strategies in agriculture, where its PrimeRoot technology combines prime editing with recombinases to insert large DNA fragments into plant genomes.
Buchholz, who serves as a scientific adviser to Seamless, plans to follow the research pointing toward a therapeutic role for his genome editor in MECP2 duplication syndrome, noting that his lab has identified an undisclosed collaboration partner contributing induced pluripotent stem cells from affected patients.
Buchholz added that longer term, he also wants to extend the approach to other DNA binding domains and other genome editing enzymes, such as transposases.
"If we could direct a transposon, for instance, into a particular locus and then only have it integrate there," he said, "this would be something that would be very valuable and very useful."