Modified Prime Editor Design for Efficient, Low-Indel Genomic Editing
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Solution Overview
Problem
Existing prime editing technologies face challenges in increasing the specificity and efficiency of genomic DNA sequence modifications, particularly in the incorporation of edited DNA strands, and are prone to indel byproducts.
Innovation Solution
Development of improved prime editor systems with engineered Cas9 and reverse transcriptase domains, including fusion proteins and uncoupled components, optimized for enhanced editing efficiency and reduced indel formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing prime editing technologies are used, then genomic DNA sequence modifications can be achieved, but editing efficiency is low and indel byproducts are frequent
Solution Approach 1:
The prime editor system is divided into separate functional modules: a Cas9 nickase domain (with H840A mutation to reduce off-target effects), a reverse transcriptase domain, and a pegRNA. This segmentation allows independent optimization of each component to improve editing efficiency while reducing indel formation through controlled nicking rather than double-strand breaks
Solution Approach 2:
The invention optimizes multiple parameters including the Cas9 nickase fidelity (H840A mutation), reverse transcriptase processivity, pegRNA structure (with extended primer binding site and poly-T tract), and editing window design. These parameter changes collectively enhance editing efficiency while minimizing indel byproducts through precise control of the editing process
2Productivity
If prime editing is performed with standard systems, then DNA strand editing can occur, but incorporation efficiency of edited DNA strands is poor
Solution Approach 1:
The system performs preliminary nicking of the non-target strand by Cas9 nickase before reverse transcription occurs. This preliminary action creates a 3' overhang that facilitates subsequent strand displacement and incorporation of the edited strand, significantly improving incorporation efficiency while maintaining precision through the controlled sequential mechanism
Solution Approach 2:
The reverse transcriptase acts as an intermediary that synthesizes a cDNA copy of the edited sequence from the pegRNA template. This intermediary step allows precise control over the edited sequence incorporation, enabling high-fidelity editing by mediating between the guide RNA and the target DNA without requiring direct DNA-DNA hybridization
Data Source
AI summary
The present disclosure provides compositions and methods for prime editing with improved editing efficiency and/or reduced indel formation with modified prime editors and prime editor fusion proteins. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure.