Prime editing
Surface model of SpCas9–reverse transcriptase–pegRNA–target DNA complex. The prime editor, composed of a SpCas9 and a reverse transcriptase, reverse transcribes template sequence in pegRNA, resulting in incorporation of desired edits to the targeted site in the genome. The RNA–DNA heteroduplex forms along the RuvC nuclease domain of SpCas9. [Shuto et al 2024]

Some researchers in the genome editing field have turned their attention to the development of tools that are not hindered by the limitations of the CRISPR-Cas9 system, such as questionable accuracy and other safety concerns.

One recently developed tool is the prime editing system, a molecule complex consisting of two components: the prime editor which combines a SpCas9 protein and a reverse transcriptase, and the prime editing guide RNA (pegRNA)—a modified guide RNA that identifies the target sequence within the DNA and encodes the desired edit.

Prime editing has already been successfully implemented in living cells of plants, zebrafish, and mice. However, due to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood.

Now, using cryo-electron microscopy, researchers at the University of Tokyo determined the spatial structure of the SpCas9–M-MLV RTΔRNaseH–pegRNA–target DNA complex in multiple states.

This work is published in Nature in the paper, “Structural basis for pegRNA-guided reverse transcription by the prime editor.

“We found the prime editor complex to be unstable under experimental conditions,” explained Yutaro Shuto, PhD, from the department of biological sciences at the University of Tokyo. “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.”

Functional analysis based on these structures also revealed how a prime editor could achieve reverse transcription without cutting both strands of the double helix. Clarifying these molecular mechanisms contributes greatly to designing gene-editing tools accurate enough for gene therapy treatments.

The researchers succeeded in determining the three-dimensional structure of the prime editor complex in multiple states during reverse transcription on the target DNA. The structures revealed that the reverse transcriptase bound to the RNA–DNA complex that formed along the part of the Cas9 protein associated with DNA cleavage, the splitting of a single strand of the double helix.

While performing the reverse transcription, the reverse transcriptase maintained its position relative to the Cas9 protein. The structural and biochemical analyses also indicated that the reverse transcriptase could lead to additional, undesired insertions.

More specifically, the authors wrote, “The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation, and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA–synthesized DNA heteroduplex builds up along the surface of SpCas9.”

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

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