Modified Prime Editing Guide RNAs for Higher Editing Efficiency
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Solution Overview
Problem
There is a need for improved prime editing guide RNAs (PEgRNAs) with enhanced efficiency for nucleotide substitutions, insertions, and deletions in DNA to correct disease-associated gene mutations.
Innovation Solution
The development of PEgRNAs comprising specific structural components such as a spacer, guide RNA core, editing template, primer binding site, and 3′ nucleic acid motifs like G-quadruplex, C-quadruplex, pseudoknot, MS2 protein binding sequence, or MMLV reverse transcriptase recruitment sequence, which enhance the editing efficiency when used with Cas proteins and DNA polymerases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PEgRNAs are used for prime editing, then basic editing function is achieved, but editing efficiency is limited
Solution Approach 1:
The PEgRNA is divided into distinct functional modules: spacer region for target recognition, guide RNA core for Cas protein binding, editing template for desired sequence, primer binding site for reverse transcription initiation, and 3' nucleic acid motifs for stability enhancement. This segmentation allows each component to be optimized independently for its specific function while working together to achieve high editing efficiency.
Solution Approach 2:
The PEgRNA employs composite nucleic acid structures combining different sequence motifs and secondary structures (such as G-quadruplexes, pseudoknots, or other stabilizing elements) at the 3' end to enhance overall stability and editing efficiency. This composite approach integrates multiple functional properties into a single molecular entity.
2Reliability
If PEgRNA stability is increased through structural modifications, then editing efficiency improves, but design and synthesis complexity increases
Solution Approach 1:
The stability of PEgRNA is enhanced by modifying specific parameters such as adding 3' nucleic acid motifs that form stable secondary structures (G-quadruplexes, pseudoknots), optimizing the length and sequence of the spacer region, and adjusting the composition of the editing template. These parameter changes increase stability without requiring complete redesign of the entire molecule.
Solution Approach 2:
Stability-enhancing 3' nucleic acid motifs are incorporated into the PEgRNA design during the synthesis planning stage. These motifs are pre-designed and integrated into the overall structure before final synthesis, allowing for streamlined manufacturing processes rather than requiring post-synthesis modifications.
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
The modified PEgRNAs demonstrate up to 2.5-fold higher editing efficiency compared to conventional PEgRNAs, facilitating precise genetic corrections.
Implementation Method 1
a spacer that comprises a region of complementarity to a search target sequence in a target strand of a double stranded target DNA
Implementation Method 2
a guide RNA (gRNA) core capable of binding to a Cas protein
Implementation Method 3
a primer binding site (PBS) that comprises a region of complementarity to a region upstream of a nick site in a non-target strand of the double stranded target DNA
Data Source
AI summary
Provided herein are compositions and methods related to modified prime editing guide RNAs.