PEgRNA-Guided Prime Editing for Precise Genome Changes Without DSBs
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR/Cas systems, face challenges in precision editing of single nucleotide mutations, including bystander editing, target nucleotide product mixtures, and limitations on insertion or deletion modifications, particularly in non-dividing human 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, enabling precise installation of single nucleotide changes or small insertions/deletions without double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homology directed repair (HDR) is used for precision editing, then editing precision is improved, but editing efficiency deteriorates (particularly in non-dividing cells)
Solution Approach 1:
The patent introduces a specialized guide RNA (PEgRNA) as an intermediary that combines homology-directed repair precision with efficient integration. The PEgRNA contains a primer binding site, a DNA synthesis template with the desired edit, and a scaffold region that recruits the Cas9-nickase-polymerase complex, enabling precise editing without relying on cellular HDR machinery
Solution Approach 2:
The prime editing system is self-sufficient as it carries its own DNA synthesis template within the PEgRNA structure. The polymerase domain of the editor synthesizes the edited DNA sequence directly at the target site using the PEgRNA template, eliminating the need for external donor DNA templates required by traditional HDR methods
2Manufacturing precision
If traditional HDR with donor DNA templates is used, then desired edits can be introduced, but indel formation increases due to competing non-homologous end joining (NHEJ)
Solution Approach 1:
The patent extracts the DNA synthesis template from external donor DNA molecules and integrates it directly into the guide RNA structure. This eliminates the competition between NHEJ and HDR pathways by providing a built-in template that the polymerase can use directly, preventing NHEJ-mediated indel formation while ensuring precise editing
Solution Approach 2:
The patent replaces the mechanical system of double-strand break formation and donor DNA integration with a nicking-based system. The Cas9-nickase creates a single-strand nick, and the polymerase extends from this nick using the PEgRNA template, substituting the complex HDR/NHEJ competition with a streamlined nick-and-extend mechanism that prevents indel formation
3Adaptability or versatility
If double strand breaks are generated for editing, then DNA repair mechanisms are activated, but chromosomal rearrangements and deletions occur
Solution Approach 1:
The patent inverts the traditional approach by instead of creating double-strand breaks to activate repair, it creates a single-strand nick and uses the undamaged complementary strand as a template for repair. The polymerase extends from the nick using the intact complementary strand and the PEgRNA template, avoiding the chromosomal damage associated with DSBs while still activating precise repair mechanisms
4Adaptability or versatility
If double strand breaks are generated for editing, then DNA repair is initiated, but cell growth arrest and apoptosis are activated through p53 axis
Solution Approach 1:
The patent inverts the traditional DSB-based editing approach by using single-strand nicks instead. This inverted approach initiates DNA repair through the same cellular machinery (avoiding the need for exogenous donor DNA integration) but does so in a way that does not trigger the p53-mediated growth arrest and apoptosis pathways, as single-strand nicks are far less genotoxic than double-strand breaks
5Productivity
If base editing is used for high efficiency editing, then editing efficiency is improved, but bystander editing of non-target bases occurs
Solution Approach 1:
The patent applies local quality by restricting the editing activity to a precise location defined by the PEgRNA sequence. The polymerase extends only from the nick site using the PEgRNA template, and the PEgRNA itself is designed with a specific spacer sequence that targets only the desired locus. This localized approach ensures high efficiency editing at the target site without bystander editing of adjacent bases, as the editing window is precisely defined by the PEgRNA structure rather than a broad deaminase activity window
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, allowing for precise installation of desired nucleotide changes and modifications, overcoming limitations of traditional methods like CRISPR/Cas systems.
Implementation Method 1
a reverse transcriptase to synthesize the single-strand DNA flap from the extension arm
Implementation Method 2
a Cas9 nickase to introduce a single-strand nick in the target DNA sequence
Data Source
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.