Chimeric Polypeptide Fusion for Genome Editing Efficiency
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Solution Overview
Problem
Current genome editing tools, such as CRISPR/Cas systems, face low efficiency in mutagenesis due to the dominance of error-free repair mechanisms over error-prone repair pathways, limiting the mutation rate and homologous recombination efficiency.
Innovation Solution
Development of chimeric polypeptides that fuse sequence-specific endonucleases with DNA modifying enzymes like RecE, RecJ, RecBCD, Mungbean nuclease, ExoI, ExoIII, ExoVII, and terminal deoxyribonucleotidyl transferase, which enhance error-prone non-homologous end joining (NHEJ) and homologous recombination by generating 3' OH overhangs or exposing recessed 3' OH at double-strand break sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If genome editing enzymes are used to create double-strand breaks, then DNA modification is achieved, but mutagenesis efficiency is low due to dominance of error-free repair pathways
Solution Approach 1:
The patent merges sequence-specific endonucleases with DNA modifying enzymes (RecE, RecJ, RecBCD, Mungbean nuclease, ExoI, ExoIII, ExoVII, TdT) to create chimeric polypeptides. This combination allows the system to simultaneously generate double-strand breaks and promote error-prone repair pathways, thereby increasing mutagenesis efficiency while reducing the dominance of error-free repair mechanisms.
Solution Approach 2:
The DNA modifying enzymes in the chimeric polypeptides act as intermediaries that facilitate error-prone non-homologous end joining and homologous recombination. These enzymes modify the DNA ends after the initial break, creating conditions that favor mutation generation and increase the overall mutagenesis efficiency of the genome editing system.
2Productivity
If chimeric polypeptides are used to enhance error-prone NHEJ, then mutation rate increases, but off-target effects may increase
Solution Approach 1:
The chimeric polypeptide is segmented into distinct functional domains: a sequence-specific endonuclease domain for targeted DNA recognition and cleavage, and a DNA modifying enzyme domain for promoting error-prone repair. This segmentation allows the system to maintain sequence specificity through the endonuclease while the DNA modifying enzyme enhances mutation rate through localized chemical modification of DNA ends.
Solution Approach 2:
The DNA modifying enzyme activity is localized to the site of double-strand breaks through the spatial coupling of the endonuclease and modifying enzyme in the chimeric polypeptide. This local quality ensures that error-prone repair is promoted specifically at target locations while minimizing off-target effects through the sequence-specificity of the endonuclease domain.
3Productivity
If RecE domain is used to generate 3' OH overhangs, then homologous recombination is enhanced, but enzyme complexity increases
Solution Approach 1:
The RecE domain is merged with the sequence-specific endonuclease in a chimeric polypeptide structure. This merging allows the RecE domain to generate 3' OH overhangs at the site of double-strand breaks, thereby enhancing homologous recombination efficiency, while the fused endonuclease provides the necessary sequence-specific targeting capability.
Solution Approach 2:
The chimeric polypeptide exhibits multi-functionality by combining the DNA cleavage function of the sequence-specific endonuclease with the DNA modification function of the RecE domain. This universal system can therefore perform both targeted break generation and promotion of homologous recombination through a single integrated enzyme complex.
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
The fusion of these enzymes increases the efficiency of genome editing by enhancing mutation and homologous recombination rates, potentially up to 30-40% in stable transformations, and up to 46% in protoplast experiments, while reducing off-target effects.
Implementation Method 1
a DNA modifying enzyme comprising a RecE domain... generates 3' OH overhangs or exposes recessed 3' OH at double-strand break sites
Data Source
AI summary
The present disclosure provides compositions and methods for increasing mutation efficiency and homologous recombination rates of site-specific endonucleases. The compositions and methods comprise a chimeric polypeptide comprising a site-specific endonuclease or a domain thereof and a functional moiety. The current inventions relate to functional enhancement of the CRISPR-Cas enzymes. Disclosed herein include possible variants and their intended improvements.