Targeted Genome Mutagenesis Using Nickase-Helicase Base Editing
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Solution Overview
Problem
Existing tools for targeted mutagenesis in the endogenous mammalian genome are limited by sensitivity to gene dosage, require exogenous overexpression, introduce genome-wide mutations, or are confined to narrow genomic windows, and can lead to artificial variants linkage.
Innovation Solution
A composition comprising a programmable nickase, helicase, and deaminase system that introduces targeted single-strand nicks, unwinds DNA, and introduces base edits, enabling continuous mutagenesis with modular components for specific genomic regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing tools require exogenous overexpression of the gene of interest on a plasmid or vector, then mutagenesis can be performed, but sensitivity to gene dosage occurs and cannot be used to evolve noncoding regions in their native chromatin contexts
Solution Approach 1:
The invention extracts the mutagenesis function from exogenous plasmid/vector systems and implements it directly in the endogenous genome through CRISPR-Cas9 mediated homology-directed repair, eliminating gene dosage sensitivity and enabling study of native chromatin contexts
Solution Approach 2:
The invention uses HDR templates as intermediaries to deliver desired mutations directly to the endogenous genome, replacing the need for exogenous gene overexpression systems and enabling precise mutagenesis in native chromatin environments
2Manufacturing precision
If tools require integrating exogenous sequences into the genome, then mutagenesis can be achieved, but experimental complexity increases and throughput is constrained
Solution Approach 1:
The invention removes the need for complex exogenous sequence integration by using small HDR templates that can be delivered as oligonucleotides, dramatically simplifying the experimental workflow while maintaining precise mutagenesis capability
Solution Approach 2:
The invention uses transient HDR templates (oligonucleotides) instead of stable integrated exogenous sequences, allowing precise mutagenesis without permanent genomic integration and reducing experimental complexity
3Area of stationary object
If existing tools are non-specific such as alkylators that introduce genome-wide mutations, then mutagenesis coverage is comprehensive, but targeting precision is lost
Solution Approach 1:
The invention applies local quality by using CRISPR-Cas9 guide RNA to direct mutagenesis to specific genomic loci, achieving both targeted precision and the ability to cover multiple locations through multiplexing, unlike non-specific alkylators
4Measurement precision
If tools are confined to narrow genomic windows such as CRISPR base-editors, then targeting precision is high, but the base positions that can be targeted with high efficiency are limited and artificial variants linkage occurs
Solution Approach 1:
The invention uses dynamic HDR template design with variable homology arm lengths and positions to adapt to different genomic contexts and target distances from the CRISPR cut site, expanding the range of targetable positions beyond fixed-window base editors
Solution Approach 2:
The invention changes parameters including homology arm length, template position, and Cas9 nicking strategy to optimize targeting of different genomic positions, overcoming the fixed-window limitation of base editors and reducing artificial variant linkage
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
Enables systematic mutagenesis across large genomic regions with tunable mutation rates, identifying functional variants and resistance to therapeutic agents, and probing non-coding gene regulation, overcoming limitations of existing tools.
Implementation Method 1
a programmable nickase configured to introduce a single-strand nick in double-stranded DNA (dsDNA) at one or more targeted nick sites
Implementation Method 2
a helicase configured to unwind a portion of the dsDNA at the one or more targeted nick sites
Implementation Method 3
a deaminase configured to introduce one or more base edits within the portion of unwound dsDNA
Data Source
AI summary
The disclosure provides for compositions, systems, and methods for long-range targeted mutagenesis. In particular, the disclosure provides engineered compositions comprising a programmable nickase configured to introduce a single-strand nick in double-stranded DNA (dsDNA) at one or more targeted nick sites; a helicase configured to unwind a portion of the dsDNA at the one or more targeted nick sites; and a deaminase configured to introduce one or more base edits within the portion of unwound dsDNA. Also provided are vector and delivery systems comprising one or more polynucleotides encoding the components of the compositions, as well as modified cells, cell populations, animal models, pharmaceutical compositions, and kits comprising the compositions.