PRIME-Del Genome Editing via Dual Strand Nicking
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Solution Overview
Problem
Current methods for precise genomic deletions using CRISPR-Cas9 technologies are limited by low precision, cytotoxicity, and the inability to program arbitrary deletions, especially for larger sequences, due to limitations in the distribution of protospacer-adjacent motifs (PAM) and the generation of unintended mutations.
Innovation Solution
The PRIME-Del method employs a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, allowing for precise deletion of sequences up to 10 kb by creating single-stranded breaks and using reverse transcriptase to generate 3' overhangs that guide DNA repair, enabling precise excision and incorporation of sequences without the need for double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 paired sgRNAs are used to induce genomic deletions, then deletion capability is achieved, but precision deteriorates due to short indels and unintended mutations
Solution Approach 1:
The patent segments the deletion process into two independent nicking events on opposite DNA strands, each mediated by a separate pegRNA-editor complex. This segmentation allows precise control over the boundaries of the deletion without generating indels at the junctions, as each nick is cleanly processed and the intervening sequence is excised as a discrete fragment.
Solution Approach 2:
The patent introduces an RNA intermediary (pegRNA) that serves as both the targeting guide and the template for repair. The pegRNA contains the spacer sequence that defines the deletion boundaries and the extended 3' tail that provides the repair template, acting as an intermediary that directs the nickase to the precise location and specifies the exact repair outcome without requiring PAM sequences.
2Productivity
If CRISPR-Cas9 induces double-strand breaks for deletion, then deletion efficiency is improved, but cytotoxicity increases
Solution Approach 1:
The patent inverts the conventional approach by using two single-strand nicks instead of one double-strand break. Each pegRNA guides a nickase to create a nick on a specific strand at a specific location. The combination of two nicks on opposite strands creates a clean double-strand break at the intended deletion boundary without the collateral damage of conventional Cas9 cleavage, significantly reducing cytotoxicity while maintaining deletion efficiency.
3Ease of manufacture
If conventional CRISPR-Cas9 deletion method is used, then simple deletion is achieved, but adaptability deteriorates due to PAM site distribution limitations
Solution Approach 1:
The patent changes the fundamental parameter of target recognition from DNA-based (PAM sequences) to RNA-based (spacer sequences in pegRNA). By using RNA spacers that are complementary to the target DNA sequence, the system can target any genomic location without being constrained by the distribution of PAM sites. The pegRNA design parameters (spacer sequence, 3' tail length, homology region) can be adjusted to achieve any desired deletion outcome, providing complete adaptability while maintaining simplicity through standardized pegRNA construction.
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-Del achieves higher precision and efficiency in genomic deletions compared to CRISPR-Cas9, allowing for concurrent deletions and insertions, and avoids the limitations of PAM site distribution, reducing unintended mutations and cytotoxicity.
Implementation Method 1
the functional reverse transcriptase domain of the first editing complex to generate a first 3′ overhang from the first single-stranded break using the first extended domain as template
Data Source
AI summary
Disclosed are methods and related compositions for genomic editing. In one aspect, methods of editing double stranded DNA (dsDNA) use first and second editing complexes specific for first and second target sequences on the sense and antisense strands of the dsDNA molecule, respectively. Each editing complex comprises an extended guide RNA associated with a fusion editor protein, which comprises a functional nickase domain and a functional reverse transcriptase domain. The respective guide RNAs guide their associated fusion editor proteins to the dsDNA, which implement single stranded breaks on opposite strands of the dsDNA. The respective reverse transcriptase domains generate 3′ overhangs. Repair of the dsDNA excises the portion of dsDNA disposed between the two single-stranded breaks. A variety of configurations and applications of the method are disclosed, providing flexible, facile, efficient, and precise methods to impose genetic manipulations.