Prime Editing Guide RNA for Precise HDR-Free Genome Writing
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Solution Overview
Problem
Current genome editing technologies face challenges in precision editing of single nucleotide mutations, including low efficiency in homology directed repair (HDR), generation of chromosomal rearrangements, and limitations of base editors such as bystander editing and target nucleotide product mixtures, limiting their therapeutic potential.
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 precise installation of single nucleotide changes and small insertions or deletions without creating double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homology directed repair (HDR) is used for precision editing, then single base pair editing capability is improved, but editing efficiency in human cell types particularly non-dividing cells deteriorates
Solution Approach 1:
The invention extracts and eliminates the requirement for homology directed repair (HDR) from the editing process. By using a prime editing guide RNA (PEgRNA) with a primer binding site and a reverse transcription template, the system enables direct reverse transcription at the target site without relying on HDR, thereby maintaining precision while dramatically improving efficiency in human cells including non-dividing cells
Solution Approach 2:
The invention introduces a reverse transcription template within the PEgRNA as an intermediary element. This template serves as a mediator that allows the polymerase to directly synthesize the edited sequence at the target site, bridging the gap between CRISPR targeting and precise editing without requiring HDR or donor DNA templates
2Adaptability or versatility
If CRISPR/Cas systems create double strand breaks for editing, then target site accessibility is improved, but chromosomal rearrangements and cell growth arrest are increased
Solution Approach 1:
The invention converts the harmful effect of double strand breaks into a beneficial process by using a nickase variant that creates only single-strand nicks. The PEgRNA design includes a primer binding site that allows the nicked strand to serve as a primer for reverse transcription, transforming the potentially harmful nick into a useful starting point for precise editing without triggering chromosomal rearrangements or p53-mediated growth arrest
Solution Approach 2:
The invention replaces the mechanical system of double strand break creation with a biochemical system based on reverse transcription. Instead of using Cas9 nuclease to create DSBs, the system uses a nickase with a PEgRNA that contains a reverse transcription template, allowing the polymerase to directly write the edited sequence at the target site through reverse transcription, thereby eliminating the harmful mechanical disruption of DNA
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 desired nucleotide changes and modifications, expanding the scope of therapeutic applications in genetic disorders.
Implementation Method 1
a reverse transcriptase, and an extended guide RNA, wherein the reverse transcriptase synthesizes a single-strand DNA flap using the extended guide RNA as a template and the target DNA as a primer, thereby incorporating the desired nucleotide change
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.