Summary
A bioRxiv preprint describes a prime-editing method that creates genomic deletions while adding molecular tags, letting researchers identify edited cells and measure gene-expression effects. In K562 cells, the team reports intronic enhancer deletions associated with 20–30% lower host-gene expression.
A research team has developed a prime-editing approach that makes targeted genomic deletions while adding molecular tags to the edited DNA. In a bioRxiv preprint, the researchers report using the method in K562 cells to study candidate regulatory elements and connect specific deletions with changes in gene expression.
The method, called OPTIMuS PRIME-Del, is designed to address two challenges in genetic screens. Conventional CRISPR knockout screens often use small insertions or deletions to disrupt a gene’s protein-coding sequence. Those small edits are less reliable for testing enhancers and other regulatory DNA, where a small change may leave the element’s activity intact. Such screens can also infer which cells were edited from the guide RNA detected in each cell, rather than directly identifying a successful edit.
Deletions carry their own molecular tags
OPTIMuS PRIME-Del uses paired prime editing to remove a chosen DNA segment and insert both a T7 phage polymerase promoter and a short barcode at the deletion site. The researchers designed pairs of prime-editing guide RNAs to program these tagged deletions. The barcode and promoter are intended to make successful edits easier to identify and quantify, rather than relying only on guide-RNA detection.
The team tested the approach on about 1,200 intronic candidate cis-regulatory elements, or cCREs, in K562 cells. Cis-regulatory elements are stretches of DNA that can influence the activity of genes on the same chromosome; an intronic element lies within a gene. The researchers also conducted a secondary screen of about 100 of the cCREs that were most editable.
Single-cell measurements link edits to gene activity
The researchers combined the editing method with single-cell RNA sequencing. This allowed them to identify cells carrying particular deletions and measure the RNA in those cells, providing a way to examine the transcriptional consequences of specific edits.
They report that deleting some intronic enhancers reduced expression of their host genes by 20–30%. The study also identified a candidate enhancer–gene interaction spanning 124 kilobases. The team further assessed these findings using sequence-based predictions of how the deletions would alter chromatin accessibility—the degree to which DNA is accessible to the molecular machinery that reads it.
The approach offers a way to test many noncoding DNA elements while linking the edit itself to measurements in individual cells. That matters because regulatory DNA can affect gene activity without changing a gene’s protein-coding sequence, and the effects of one such element may be difficult to distinguish from those of another in pooled screens.
The results are a cell-based proof of concept reported in a preprint. They demonstrate the method in K562 cells and concern the functional study of regulatory DNA, rather than a therapeutic application.