Genome Editing With RT-DBD Nuclease for Site-Specific Insertions
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Solution Overview
Problem
Existing methods for integrating nucleic acid sequences into a genome lack site specificity and efficiency, particularly for longer sequences, and often require multiple steps or rely on host repair pathways.
Innovation Solution
A system comprising a polypeptide with a reverse transcriptase (RT) domain, DNA-binding domain (DBD), and endonuclease domain, along with a template RNA, is used to specifically target and modify DNA by inserting, altering, or deleting sequences, utilizing a heterologous targeting domain and homology domains for precise genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR/Cas9 is used for genome editing, then small edits can be achieved, but integration of longer sequences is less effective
Solution Approach 1:
The system divides the genome editing function into separate domains: a DNA-binding domain for target recognition, an endonuclease domain for DNA cleavage, and a reverse transcriptase domain for template-directed synthesis. This segmentation allows each domain to optimize its function independently, enabling both precise targeting and efficient integration of longer sequences.
Solution Approach 2:
The patent introduces a template RNA as an intermediary that carries the sequence to be integrated. The reverse transcriptase domain uses this RNA template to synthesize DNA that is then integrated into the genome at the target site, mediating between the binding/cleavage functions and the final integration outcome.
2Productivity
If Cre/loxP approach is used, then sequence insertion can be achieved, but it requires multiple steps (first inserting loxP site, then inserting sequence of interest)
Solution Approach 1:
The system merges multiple functions into a single polypeptide: target site binding, DNA strand cleavage, and template-directed DNA synthesis are combined in one protein complex. This allows all steps of sequence insertion to occur in a single coordinated action rather than requiring separate sequential steps.
Solution Approach 2:
The polypeptide is designed with multi-functionality, where the same protein complex performs binding, cleavage, and synthesis functions. The DNA-binding domain provides universality in target recognition, while the reverse transcriptase domain provides universality in template-directed synthesis, making the system applicable to various insertion scenarios.
3Device complexity
If nucleic acid integration is performed without specialized proteins, then the process is simple, but integration occurs at low frequency with little site specificity
Solution Approach 1:
The DNA-binding domain provides local quality in terms of target site recognition, with specific amino acid sequences that recognize and bind to particular DNA sequences. This localized binding capability confers high site specificity to the integration process without requiring complex multi-component systems.
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 system enables efficient and site-specific insertion or deletion of nucleic acid sequences into the genome, achieving precise modifications with high accuracy and efficiency.
Implementation Method 1
a polypeptide with a reverse transcriptase (RT) domain, DNA-binding domain (DBD), and endonuclease domain
Implementation Method 2
an endonuclease domain, e.g., a nickase domain
Implementation Method 3
a DNA-binding domain (DBD)... a heterologous targeting domain that binds specifically to a sequence comprised in the target site
Implementation Method 4
a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence (e.g., a CRISPR spacer) that binds a target site
Data Source
AI summary
Methods and compositions for modulating a target genome are disclosed. This disclosure relates to novel compositions, systems and methods for altering a genome at one or more locations in a host cell, tissue or subject, in vivo or in vitro. In particular, the invention features compositions, systems and methods for inserting, altering, or deleting sequences of interest in a host genome.