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Probing the Inner World of Cells Provides Unexpected Boost to Prime Editing

An image showing the words "prime editing".
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Prime editing is the “new kid on the block” for genome editing, offering scientists precise control over how genome sequences are changed. Though novel, the technique has already been used widely in cancer research, to restore normal function in sickle cell disease patient’s cells and to efficiently insert large DNA segments in plants, among other applications.

"Prime editing is such an incredibly powerful genome-editing tool,” said Dr. Britt Adamson, assistant professor of molecular biology at Princeton University.

Still in its infancy compared to other more established genome-editing techniques, prime editing has one shortfall; while it’s incredibly precise and versatile, it can be slow, producing edits at a low efficiency.

Adamson and colleagues at Princeton have been exploring different approaches to tackle this drawback. In Nature, they share a new, more efficient prime editor.

Do unknown cellular processes aid or hinder prime editing?

Prime editors are molecular machines that consist of an engineered Cas9 variant that is fused to a reverse transcriptase enzyme. The Cas9 element is guided to the target site by a prime editing guide RNA, or pegRNA, which enables sequence-specific DNA binding and nickase activity, while the reverse transcriptase produces a DNA molecule encoding a desired mutation.

Adamson and Jun Yan, a graduate student in Adamson’s research group, hypothesized that unknown processes occurring in cells could potentially hinder or accelerate prime editing. “To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference (CRISPRi) screens,” the researchers explained.

What is a CRISPRi screen?

A library of guide RNAs (gRNAs) targeting different genes is introduced to cells, along with the CRISPRi system, a modified version of the CRISPR-Cas9 system that enables precise control of gene expression, without affecting the DNA sequence. The gRNAs direct the Cas9-repressor complex to a target gene where it interferes with transcription, thereby reducing or completely silencing gene expression. This allows researchers to explore the effects of gene knockdown on cellular phenotypes.

Yan decided that he would first engineer a cell line that emits green fluorescence when specific prime edits are inserted. Next, he systematically blocked the expression of proteins that are typically expressed in those cells and measured the fluorescence to assess which of those proteins affected the prime editing process.

Yan identified 36 cellular determinants of prime editing, and only one – a small RNA-binding protein called La – promoted prime editing. Under normal physiological conditions, La binds to certain DNA sequences that are found at the ends of RNA molecules and protects them from being degraded. These sequences, known as polyuridine tracts, are a typical byproduct of pegRNA expression in cells.

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“Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts,” the research team said. “We found that La functionally interacts with the 3′ ends of pegRNAs.”

Based on these findings, Adamson and colleagues fused the part of La that binds polyuridine tracts to a prime editing protein. This protein, called PE7, had a much higher editing efficiency than standard prime editors, with very low byproducts.

Efficient prime editor tool has clinical potential

Scientists at Boston Children's Hospital, Harvard Medical School and the University of California learned of Adamson and colleagues’ results and wanted to apply PE7 in disease-relevant cells.

“We next evaluated editing with PE7 at additional genomic targets, including ones associated with sickle cell disease (HBB), prion disease (PRNP), familial hypercholesterolaemia (PCSK9), adoptive T cell transfer therapy (IL2RB), HIV infection (CXCR4) and CDKL5 deficiency disorder (CDLK5),” Adamson and colleagues described.

As with their previous experiment, editing at these genomic targets was substantially improved using PE7 compared to standard prime editors.

“Although the exact mechanism (or mechanisms) by which La promotes prime editing and the boundaries within which PE7 provides improvement remain to be fully elucidated (for example, across additional cell types, delivery modalities and editing conditions), our study represents an important first step in understanding this key cellular determinant and exploiting its function for optimization,” the researchers said.

"This work is a beautiful example of how deeply probing the inner workings of cells can lead to unexpected insights that may yield near-term biomedical impact,” Dr. Daniel Bauer, a co-author of the study and attending physician at Boston Children’s Hospital, and the Donald S. Fredrickson, MD Associate Professor of Pediatrics at Harvard Medical School, concluded.

Reference: Yan J, Oyler-Castrillo P, Ravisankar P, et al. Improving prime editing with an endogenous small RNA-binding protein. Nature. 2024;628(8008):639-647. doi:10.1038/s41586-024-07259-6

This article is a rework of a press release issued by Princeton University. Material has been edited for length and content.