Prime Editing Fusion Proteins for Precise DNA Sequence Correction

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Solution Overview

Problem

Current genome editing technologies face challenges in achieving precise and efficient single nucleotide changes, insertion or deletion of base pairs, and are limited by bystander editing and cell-type dependent efficiencies, particularly in non-dividing cells.

Innovation Solution

The development of prime editing, which uses a nucleic acid programmable DNA binding protein (napDNAbp) in conjunction with a polymerase and a prime editing guide RNA (PEgRNA) to directly write new genetic information into a specified DNA site through target-primed reverse transcription, allowing for precise installation of desired nucleotide changes or modifications.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If homology directed repair (HDR) is used to insert or correct DNA sequences at double strand break sites, then DNA sequence correction is achieved, but editing efficiency is very low in most human cell types particularly in non-dividing cells

Engineering Contradiction:

ImproveDNA sequence correction precisionVSAvoidediting efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent introduces a prime editing guide RNA (PEgRNA) as an intermediary that combines features of both CRISPR guide RNA and DNA repair template. The PEgRNA contains a primer binding site, spacer sequence, and edit template that together mediate the editing process without requiring donor DNA templates or creating double-strand breaks, thereby achieving both high precision and high efficiency in DNA sequence correction across all cell types including non-dividing cells

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces the mechanical system of double-strand break creation and homology-directed repair with a biochemical system based on reverse transcription. Instead of using Cas9 nuclease to create DSBs followed by HDR, the system uses a prime editor fusion protein with reverse transcriptase activity that directly synthesizes the edited sequence onto the PEgRNA template, substituting a more efficient biochemical pathway for the less efficient mechanical repair process

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Productivity

If base editing is used to edit target nucleotides without creating double strand breaks, then editing efficiency is high, but bystander editing of non-target bases occurs and target bases must be located at specific positions relative to PAM sequence

Engineering Contradiction:

Improveediting efficiencyVSAvoidediting precision

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent applies local quality by designing the PEgRNA edit template to contain only the specific nucleotide changes desired at the target site, rather than affecting all bases within a window. The reverse transcription process copies only the intended edits from the PEgRNA template, ensuring that precision is maintained while efficiency remains high, as the editing is localized exactly where the PEgRNA hybridizes to the target DNA

Inventive Principle:

Principle #3Local quality

3Adaptability or versatility

If traditional CRISPR-Cas systems are used to access genomic sites, then nearly any conceivable genomic site can be accessed, but precision editing of single base pairs remains a major challenge

Engineering Contradiction:

Improvegenomic site accessibilityVSAvoidsingle base pair editing precision

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The patent merges multiple functions into a single prime editor fusion protein and PEgRNA system. The PEgRNA combines the targeting function (spacer sequence), the programming function (edit template with desired changes), and the primer function (primer binding site) into one molecule. This unified system maintains the versatility of CRISPR for accessing any genomic site while achieving precise single base pair editing through the integrated reverse transcription process

Inventive Principle:

Principle #5Merging (Combining)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Prime editing achieves high efficiency and flexibility in genome editing, enabling precise installation of single nucleotide changes, insertions, or deletions with reduced off-target effects and cell-type dependency.

Implementation Method 1

a reverse transcriptase, and a prime editing guide RNA (PEgRNA)... to directly write new genetic information into a specified DNA site through target-primed reverse transcription

Methodology Applied

Scientific EffectReverse transcription:

Data Source

PatentUS12570972B2Methods and compositions for prime editing nucleotide sequences

Publication Date: 2026.03.10 THE BROAD INST INC

AI summary

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.