Prime Editing via Single-Strand Nicks
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Solution Overview
Problem
Current gene editing technologies, such as CRISPR-Cas9, rely on double-strand breaks which can lead to undesirable outcomes like chromosomal translocations and insertion/deletion events, and are limited in their ability to perform all necessary point mutations, particularly transversion mutations, and are constrained to editing near the protospacer adjacent motif (PAM).
Innovation Solution
The use of nucleic acid programmable DNA binding proteins with nickase activity, such as nCas9, combined with a DNA polymerase and a chimeric prime editing guide polynucleotide, allows for precise editing of double-stranded DNA by creating a nick and synthesizing new DNA strands to incorporate intended nucleotide edits without inducing double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If programmable nucleases (CRISPR-Cas9) are used to form double-strand breaks for genome editing, then insertion and deletion of genomic DNA can be achieved, but translocations of chromosomes, p53 activation, and undesirable insertion/deletion events occur
Solution Approach 1:
The invention divides the double-strand break function into two separate single-strand nicking functions. Instead of using one nuclease to cut both strands simultaneously, two separate nickases are used: one nickase creates a nick on the first strand, and another nickase creates a nick on the second strand at a different location. This segmentation eliminates the harmful effects of DSBs while maintaining genome editing capability through controlled single-strand nicks that are repaired by the cell's natural base excision repair pathway.
2Manufacturing precision
If base editors are used for precision base editing, then transition mutations can be achieved, but transversion mutations necessary to treat all human genetic diseases cannot be performed
Solution Approach 1:
The invention creates a universal nickase-based editing platform that can perform all types of point mutations (both transitions and transversions) through a single mechanism. By using programmable nickases with guide RNAs that can target any DNA sequence, the system achieves multi-functionality comparable to that of programmable endonucleases, while avoiding DSB-related harms. The nickase mechanism universally handles all mutation types through controlled single-strand nicks and cellular repair pathways.
3Measurement precision
If programmable endonucleases or base editors are used for editing, then editing can be performed at targeted locations, but editing is constrained to a narrow region proximal to the PAM
Solution Approach 1:
The invention inverts the conventional approach by placing the PAM requirement on the non-target strand rather than the target strand. Traditional CRISPR systems require the PAM to be adjacent to the target strand for recognition and cutting. The nickase system uses guide RNAs that bind to the non-target strand's PAM sequence, allowing the actual editing to occur at locations farther from the traditional PAM constraint. This inversion expands the editable region while maintaining precise targeting capability through guide RNA complementarity.
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 enables more versatile and precise gene editing, capable of performing a wide range of mutations, including transversions, and editing at locations farther from the PAM, reducing the risk of undesirable outcomes associated with double-strand breaks.
Implementation Method 1
a nucleic acid programmable DNA binding protein (napDNAbp) with nickase activity (e.g., nCas9)
Implementation Method 2
wherein the RNA segment hybridizes to the nicked edit strand of the double-stranded target polynucleotide
Implementation Method 3
wherein the DNA polymerase synthesizes a single stranded DNA that replaces an editing target sequence in the edit strand
Data Source
AI summary
Provided herein are prime editing systems featuring prime editors complexed with a chimeric prime editing (PE) guide polynucleotide. Systems, prime editor fusion proteins and methods of using such editors for editing a double-stranded DNA target sequence are also provided.