Prime editing systems (PEs) hold great promise in modern biotechnology. However, their editing range is limited as PEs can only modify the downstream sequences of the pegRNA nick. Here, we report the development of the extended prime editor system (EXPERT) to overcome this limitation by using an extended pegRNA (ext-pegRNA) with modified 3' extension, and an additional sgRNA (ups-sgRNA) targeting the upstream region of the ext-pegRNA. We demonstrate that EXPERT can efficiently perform editing on both sides of the ext-pegRNA nick, a task that is unattainable by canonical PEs. EXPERT exhibits prominent capacity in replacing sequences up to 88 base pairs and inserting sequences up to 100 base pairs within the upstream region of the ext-pegRNA nick. Compared to canonical PEs such as PE2, the utilization of the EXPERT strategy significantly enhances the editing efficiency for large fragment edits with an average improvement of 3.12-fold, up to 122.1 times higher. Safety wise, the use of ups-sgRNA does not increase the rates of undesirable insertions and deletions (indels), as the two nicks are on the same strand. Moreover, we do not observe increased off-target editing rates genome-wide. Our work introduces EXPERT as a PE tool with significant potential in life sciences.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11822059 | PMC |
| http://dx.doi.org/10.1038/s41467-025-56734-9 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Molecular breeding of pigs in the genome editing era.
Genet Sel Evol
March 2025
College of Animal Sciences, Jilin University, Changchun, 130062, China.
Background: To address the increasing demand for high-quality pork protein, it is essential to implement strategies that enhance diets and produce pigs with excellent production traits. Selective breeding and crossbreeding are the primary methods used for genetic improvement in modern agriculture. However, these methods face challenges due to long breeding cycles and the necessity for beneficial genetic variation associated with high-quality traits within the population.
View Article and Find Full Text PDFIn vivo prime editing rescues photoreceptor degeneration in nonsense mutant retinitis pigmentosa.
Nat Commun
March 2025
Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
The next-generation gene editing tool, prime editing (PE), is adept at correcting point mutations precisely with high editing efficiency and rare off-target events and shows promising therapeutic value in treating hereditary diseases. Retinitis pigmentosa (RP) is the most common type of inherited retinal dystrophy and is characterized by progressive degeneration of retinal photoreceptors and, consequently, visual decline. To date, effective treatments for RP are lacking.
View Article and Find Full Text PDFTBC1D30 regulates proinsulin and insulin secretion and is the target of a genomic association signal for proinsulin.
Diabetologia
March 2025
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Aims/hypothesis: Components of the insulin processing and secretion pathways remain incompletely understood. Here, we examined a genome-wide association study (GWAS) signal for plasma proinsulin levels. Lead GWAS variant rs150781447-T encodes an Arg279Cys substitution in TBC1 domain family member 30 (TBC1D30), but no role for this protein in insulin processing or secretion has been established previously.
View Article and Find Full Text PDFHuman genome sequencing efforts in healthy and diseased individuals continue to identify a broad spectrum of genetic variants associated with predisposition, progression, and therapeutic outcomes for diseases like cancer . Insights derived from these studies have significant potential to guide clinical diagnoses and treatment decisions; however, the relative importance and functional impact of most genetic variants remain poorly understood. Precision genome editing technologies like base and prime editing can be used to systematically engineer and interrogate diverse types of endogenous genetic variants in their native context .
View Article and Find Full Text PDFMouse models represent a powerful platform to study genes and variants associated with human diseases. While genome editing technologies have increased the rate and precision of model development, predicting and installing specific types of mutations in mice that mimic the native human genetic context is complicated. Computational tools can identify and align orthologous wild-type genetic sequences from different species; however, predictive modeling and engineering of equivalent mouse variants that mirror the nucleotide and/or polypeptide change effects of human variants remains challenging.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!