Prime Editing PEgRNA and Reverse Transcription for Precise DNA Changes
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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' technology, 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 or small insertions/deletions without creating double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homology directed repair (HDR) is used to correct single nucleotide mutations, then precision editing can be achieved, but editing efficiency is extremely low in most human cell types
Solution Approach 1:
The invention segments the editing process into two distinct phases: (1) creating a single-strand nick to prime reverse transcription, and (2) installing the desired nucleotide change through target-primed reverse transcription. This segmentation allows the system to avoid relying on inefficient HDR while achieving both precision and high efficiency.
Solution Approach 2:
The invention introduces an intermediary mechanism (reverse transcription) between the nicking event and the final DNA repair. The reverse transcriptase uses the nicked DNA as a primer to synthesize a new strand containing the desired edit, which then serves as a template for cellular repair mechanisms, bypassing the need for exogenous donor templates required by HDR.
2Productivity
If base editing is used to achieve high-efficiency editing, then editing efficiency is improved, but bystander editing and target nucleotide product mixtures occur
Solution Approach 1:
The invention applies local quality by designing the PEgRNA to contain a specific reverse transcription template sequence that encodes only the desired single nucleotide change. This ensures that editing is restricted to the precise target location with the exact intended outcome, preventing bystander editing and product mixtures while maintaining high efficiency.
3Adaptability or versatility
If CRISPR/Cas systems create double-strand breaks to enable editing, then editing capability is achieved, but chromosomal rearrangements and deletions occur
Solution Approach 1:
The invention inverts the conventional approach by using a nickase (single-strand cutting enzyme) instead of a nuclease that creates double-strand breaks. The single-strand nick is sufficient to prime reverse transcription and enable editing without triggering the harmful chromosomal rearrangements and deletions associated with DSBs.
4Manufacturing precision
If traditional HDR methods are used for precision editing, then single nucleotide changes can be introduced, but the process requires donor DNA repair templates and has very low efficiency
Solution Approach 1:
The invention merges the guide RNA function with the reverse transcription template function into a single PEgRNA molecule. This consolidation eliminates the need for separate donor DNA templates and simplifies the editing process while maintaining precise single nucleotide editing capability.
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, precise genome editing with flexibility to introduce any desired single nucleotide change or small modifications, overcoming limitations of existing methods and expanding therapeutic potential for genetic disorders.
Implementation Method 1
the reverse transcriptase synthesizes a single-strand DNA flap by reverse transcribing the RNA extension
Implementation Method 2
a spacer sequence that anneals to a complementary protospacer in the target DNA
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 incoporated into the target DNA molecule.