Engineered pegRNAs with Toehold Motifs for Prime Editing
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Solution Overview
Problem
Prime editing, a nucleic acid editing platform, faces challenges such as reduced affinity to nucleic acid programmable DNA binding proteins, increased susceptibility to degradation, and unwanted duplex formation between the extension arm and spacer sequence of prime editing guide RNAs (pegRNAs), leading to inefficiencies and specificity issues.
Innovation Solution
Modification of pegRNAs by appending RNA structural motifs like prequeosine1-1 riboswitch aptamer, frameshifting pseudoknots, G-quadruplexes, or hairpin structures to enhance stability and reduce unwanted interactions, along with computational algorithms to identify optimal nucleotide linkers for improved performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If pegRNA extension arm is designed to include DNA synthesis template, then prime editing capability is enabled, but unwanted duplex formation between extension arm and spacer sequence occurs
Solution Approach 1:
A toehold sequence is introduced as an intermediary element that mediates between the extension arm and spacer sequence. The toehold occludes the primer binding site to prevent unwanted duplex formation, but dissociates upon napDNAbp binding to allow proper prime editing function. This intermediary resolves the contradiction by temporarily blocking harmful interactions while permitting necessary editing activity.
Solution Approach 2:
The toehold sequence provides preliminary anti-action by preemptively blocking the primer binding site before unwanted duplex formation can occur. By occupying the primer binding site with a protective toehold structure, the system prevents harmful self-complementarity while awaiting the arrival of the napDNAbp complex, which then triggers release of the block.
2Stability of the object's composition
If pegRNA is modified with RNA structural motifs, then stability and half-life are increased, but device complexity increases
Solution Approach 1:
Chemical modifications to the pegRNA nucleotides are implemented to change the physical-chemical parameters of the RNA molecule. These modifications enhance resistance to degradation by nucleases, thereby increasing stability and half-life. The parameter changes occur at the molecular level without fundamentally altering the overall pegRNA architecture or its functional mechanisms.
3Object-generated harmful factors
If primer binding site binding affinity to spacer sequence is reduced, then unwanted duplex formation is decreased, but binding affinity to target DNA may be affected
Solution Approach 1:
The toehold sequence is designed with specific local properties that differ from the main spacer sequence. It has sequence composition and secondary structure characteristics that promote selective binding - the toehold region is optimized to bind the primer binding site with high affinity while the rest of the spacer maintains appropriate affinity for target DNA. This local differentiation allows differential binding behavior without compromising overall target recognition.
Data Source
AI summary
The disclosure provides modified pegRNAs comprising one or more appended nucleotide structural motifs which increase the editing efficiency during prime editing, increase half-life in vivo, and increase lifespan in a cell. Modifications include, but are not limited to, an aptamer (e.g., prequeosim-1 riboswitch aptamer or “evopreQi-1”) or a variant thereof, a pseudoknot (the MMLV viral genome pseudoknot or “Mpknot-1”) or a variant thereof, a tRNA (e.g., the modified tRNA used by MMLV as a primer for reverse transcription) or a variant thereof, or a G-quadruplex or a variant thereof. The disclosure further provides prime editor complexes comprising the modified pegRNAs and having improved characteristics and/or performance, including stability, improved cellular lifespan, and improved editing efficiency. The disclosure also provides methods of editing a genome using the prime editor complexes with modified pegRNAs, and to nucleotide sequences and expression vectors encoding said prime editors and modified pegRNAs, and to cells, kits, and pharmaceutical compositions comprising the improved prime editor complexes.