Structural Insights into a Novel Gene-Editing Tool

Yutaro Shuto, Ryoya Nakagawa, and Osamu Nureki of the University of Tokyo conducted collaborative research to ascertain the spatial organization of different processes within a unique gene-editing instrument known as “prime editor.”

Functional analysis of these structures also demonstrated how a “prime editor” can perform reverse transcription, synthesizing DNA from RNA, without cutting both double helix strands. Understanding these molecular processes is essential to creating gene-editing instruments precise enough for gene therapy applications. The research was published in the journal Nature.

Jennifer Doudna and Emmanuelle Charpentier won the 2020 Nobel Prize in Chemistry for creating a novel yet straightforward method of editing DNA, the “blueprint” of living things. While their discovery opened new avenues for research, concerns about the accuracy of the method and the safety of cutting both strands of DNA limited its use in gene therapy treatments. Consequently, research has been underway to develop tools that avoid these drawbacks.

One such instrument is the prime editing system, a two-component molecular complex. One part is the primary editor, which combines reverse transcriptase, an enzyme that transcribes RNA into DNA, with the SpCas9 protein, which was utilized in the original CRISPR-Cas gene editing system.

The second component is prime editing guide RNA (pegRNA), a modified guide RNA that recognizes the target sequence in the DNA and encodes the desired edit. The principal editor in this complex replaces genomic information appropriately, operating similarly to a “word processor.”

The instrument has already been effectively used in mice, zebrafish, and plants living cells. However, because its spatial organization is unknown, it is unclear exactly how this molecular complex carries out each stage of the editing process.

We became curious about how the unnatural combination of proteins Cas9 and reverse transcriptase work together.”

Yutaro Shuto, Study First Author, University of Tokyo

Cryogenic electron microscopy, an imaging method that enables observations at the near-atomic size, was employed by the research team. Further difficulties arose from the method's requirement that samples be kept in glassy ice to shield them from any harm from the electron beams.

We found the prime editor complex to be unstable under experimental conditions, so, it was very challenging to optimize the conditions for the complex to stay stable. For a long time, we could only determine the structure of Cas9.”

Yutaro Shuto, Study First Author, University of Tokyo

After overcoming numerous obstacles, the researchers were able to ascertain the prime editor complex's three-dimensional structure in each of the several states during reverse transcription on the target DNA.

The structures demonstrated that the reverse transcriptase bonded to the RNA–DNA complex that developed along the Cas9 protein's “part” that is linked to DNA cleavage, or the breaking of a double helix into a single strand. The reverse transcriptase remained concerning the Cas9 protein during the reverse transcription process. According to the structural and biochemical investigations, the reverse transcriptase may result in more unwanted insertions.

These discoveries have created new opportunities for fundamental and practical study. Shuto then outlines what comes next.

Our structure determination strategy in this study can also be applied to prime editors composed of a different Cas9 protein and reverse transcriptase. We want to utilize the newly obtained structural information to lead to the development of improved prime editors.”

Yutaro Shuto, Study First Author, University of Tokyo

Source:
Journal reference:

Shuto, Y., et al. (2024) Structural basis for pegRNA-guided reverse transcription by a prime editor. Nature. doi.org/10.1038/s41586-024-07497-8

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