Single pegRNA Template Jumping for Large DNA Insertions

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Solution Overview

Problem

Current gene editing methods, such as TwinPE, are inefficient for inserting large DNA sequences (>100 bp) without introducing double-strand breaks.

Innovation Solution

A method and system using a single pegRNA, called bidirectional pegRNA or Template-jumping Prime Editing (TJ-PE), which incorporates two primer binding sites (PBS) to enable the insertion of long DNA sequences into a target location without double-strand breaks, utilizing CRISPR/Cas DNA nuclease/nickase.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If TwinPE technology is used to insert DNA sequences, then insertion capability is improved, but insertion efficiency for large sequences (>100 bp) deteriorates

Engineering Contradiction:

Improveinsertion capabilityVSAvoidinsertion efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The pegRNA is divided into two functional segments: a first PBS for initiating reverse transcription and a second PBS for template jumping. This segmentation allows the system to handle large insertions by breaking the insertion process into manageable steps, thereby improving efficiency for large sequences while maintaining versatility

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The second PBS acts as an intermediary element that facilitates template jumping during reverse transcription. This intermediary mechanism enables efficient transfer of the reverse transcription template to the second PBS, solving the efficiency problem for large insertions while preserving the ability to insert sequences of various lengths

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If TwinPE technology is used, then large DNA deletions and replacements are enabled, but system complexity increases

Engineering Contradiction:

Improveediting functionalityVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

Multiple editing functionalities (insertions, deletions, replacements) are merged into a single prime editing system using one pegRNA with dual PBS. This consolidation maintains versatile editing capabilities while reducing system complexity compared to using separate systems for each editing type

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The single pegRNA with dual PBS is designed to perform multiple editing functions (insertions of various sizes, deletions, and replacements) through a unified mechanism. This multi-functionality achieves broad editing capability without requiring separate specialized systems, thereby reducing overall system complexity

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Manufacturing precision

If traditional prime editing is used, then precise nucleotide changes are achieved, but large sequence insertions (>100 bp) become inefficient

Engineering Contradiction:

Improveediting precisionVSAvoidinsertion efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The pegRNA system is made dynamic through the template jumping mechanism, where the reverse transcription template can be transferred to the second PBS during the editing process. This dynamic adaptation allows the system to maintain high precision for small edits while efficiently handling large insertions, resolving the contradiction between precision and efficiency

Inventive Principle:

Principle #15Dynamics

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

This approach is more cost-effective and efficient in inserting DNA sequences up to 800 bp or more, enabling precise genome editing for therapeutic applications.

Implementation Method 1

the Cas9 nickase domain binds and nicks the target genomic DNA site, which is specified by the pegRNA's spacer sequence

Methodology Applied

Scientific EffectCRISPR/Cas DNA binding and nicking:

Implementation Method 2

the reverse transcriptase domain uses the nicked genomic DNA as a primer to initiate the synthesis of an edited DNA strand using an engineered extension on the pegRNA as a template for reverse transcription

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 3

cellular DNA repair resolves the 3' flap intermediate by the displacement of a 5' flap species that occurs via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA sequence, and ligation of the new 3' flap to incorporate the edited DNA strand

Methodology Applied

Scientific EffectDNA strand invasion and displacement:

Data Source

PatentUS20250290100A1SINGLE pegRNA-MEDIATED LARGE INSERTIONS

Publication Date: 2025.09.18 UNIV OF MASSACHUSETTS

AI summary

The invention described herein provide methods and systems for deleting or inserting long (e.g., >100-500 bp) DNA sequences into a target DNA sequence using a single prime editing guide RNA (pegRNA) in conjunction with a CRISPR/Cas DNA nuclease.a