artist's illustration of DNA and genome editing

The Next Revolution in Gene Editing?

In just 10 years, CRISPR has revolutionized gene editing. The technique has fueled a wave of new scientific discoveries in cancer, Alzheimer’s, and heart disease, and several gene therapies that deploy the gene editor will soon be available to patients.

But the first-generation version of the gene-editing system—CRISPR-Cas9—does have some drawbacks. Though it allows scientists to edit the genome at a precise location, controlling the change that’s made is often difficult, and the final edit is hard to predict.

Next-generation gene editors are needed, and a promising new technology, which relies on parasitic “jumping genes,” is under development in the laboratory of Sam Sternberg, PhD, assistant professor of biochemistry & molecular biophysics, and may be able to overcome the limitations of the first generation of gene editing.

Taming jumping genes

Jumping genes—also called transposons—are mobile regions in the genome that jump randomly into other parts of the genome. Although transposons often cause harm to the organism due to this lack of specificity, they mobilize using highly efficient “integrase” enzymes that can paste large genetic payloads into the genome. The Sternberg lab previously discovered a unique family of bacterial transposons that naturally exploit CRISPR to control their specificity, and thus offer a new mode of gene editing that has both remarkable accuracy and efficient DNA insertion activity.

Sternberg and his colleagues previously demonstrated that the transposon-based gene editor appears less likely to edit unintended genomic targets than the original CRISPR technology.

One reason the new editor achieves its remarkable accuracy, the researchers report today in the journal Nature, lies in a single protein in the editing complex.

illustrations of molecules involved in gene editing

Molecular details of the TnsC protein, including its 3D structure (above), should help the researchers optimize the new gene editor for genome engineering of human cells. Image: Hoffmann, Kim, Beh et al., Nature (2022).

In the new work, Sternberg’s team, led by PhD student Florian Hoffmann and postdoctoral scientists Minjoo Kim, PhD, and Leslie Beh, PhD, used biochemical techniques to identify all the sites in the genome that the editor’s proteins bind during gene insertion in a bacterium. This approach revealed that while the editor’s main protein complex binds many off-target sites, only a small number of those sites can recruit the TnsC protein, which is essential for gene insertion.

The process operates similarly to a set of search filters a user might apply during an advanced search online. “These gene editing systems first sample a large set of possible target sites, and then TnsC is the filter that selects only the correct on-target site for DNA integration,” says Sternberg.

The researchers also revealed the 3D structure of TnsC in work led by Israel Fernández, PhD, a collaborator at St. Jude Children’s Research Hospital, which revealed that this critical protein forms a ring around the DNA and precisely positions the genomic target site for transposon insertion.

Human applications?

The new details about the molecular mechanics of these transposons as they edit bacterial genomes should help researchers optimize the new gene editors for genome engineering of human cells and clinical applications, Sternberg says.

The transposon-based editor’s ability to make large gene insertions with exquisite precision could be especially useful in certain types of gene therapy. While the original CRISPR-Cas9 tool is limited to making small alterations, the new editor could potentially insert complete, functional genes into patient cells.

Sternberg says that although clinical applications such as this will require further technological development, his team has already met one challenge and successfully adapted the gene editor to operate in human cells in the lab.


More information

The paper, titled “Selective TnsC recruitment enhances the fidelity of RNA-guided transposition,” was published on Aug. 24 in Nature.

All authors (all from Columbia except where noted): Florian T. Hoffmann, Minjoo Kim, Leslie Y. Beh, Jing Wang, Phuc Leo H. Vo, Diego R. Gelsinger, Jerrin Thomas George, Christopher Acree, Jason T. Mohabir, Israel S. Fernández (St. Jude Children’s Research Hospital), Samuel H. Sternberg.

This research was supported by grants from the Simons Foundation (SF349247), the International Human Frontier Science Program Organization, the National Institutes of Health (GM103310, DP2HG011650-01, and R01EB031935-01), a Pew Biomedical Scholarship, a Sloan Research Fellowship, a start-up package from Columbia University Vagelos College of Physicians and Surgeons, and the Vagelos Precision Medicine Fund.

Columbia University has filed patent applications related to CRISPR-transposon systems, for which S.H.S. and P.L.H.V. are listed as inventors. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits.