Genome Modulation Polypeptide for Precise Long-Sequence Insertion
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Solution Overview
Problem
Existing methods for integrating nucleic acid sequences into genomes 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 or DNA, is used to specifically target and modify genomic sequences, enabling insertion, deletion, or alteration of nucleotides with high precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing approaches like CRISPR/Cas9 are used for genome integration, then small edits can be achieved, but integration of longer sequences is less effective
Solution Approach 1:
The patent combines multiple functional domains into a single polypeptide: a reverse transcriptase domain for synthesizing DNA from RNA templates, a DNA-binding domain for target recognition, and an endonuclease domain for creating DNA breaks. This multi-functional fusion enables the system to integrate longer sequences with high precision by coordinating synthesis, targeting, and integration functions in one molecular complex.
Solution Approach 2:
The patent introduces template RNA as an intermediary carrier that bridges the gap between the polypeptide system and the target genome. The template RNA contains the heterologous sequence to be integrated and guides the polypeptide to the specific target site through base pairing, enabling precise integration of longer sequences that would otherwise be difficult to deliver and integrate.
2Manufacturing precision
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 patent merges target site recognition, DNA breaking, and sequence integration into a single simultaneous action by the multi-domain polypeptide. The DNA-binding domain locates the target site, the endonuclease domain creates a break, and the reverse transcriptase domain integrates the template sequence all in one coordinated process, eliminating the need for separate steps required by Cre/loxP methodology.
Solution Approach 2:
The template RNA is pre-loaded with the heterologous sequence and target homology information before the reaction begins. This preliminary preparation allows the system to perform insertion in a single step, as the sequence to be integrated is already positioned and ready for immediate insertion upon target site recognition and cleavage.
3Productivity
If no specialized protein is used, then genome integration can occur, but it happens at low frequency and with little site specificity
Solution Approach 1:
The patent merges three critical functions into one polypeptide: reverse transcriptase activity for DNA synthesis, DNA-binding capability for target recognition, and endonuclease activity for DNA cleavage. This combination ensures both high site specificity (through the DNA-binding domain) and high integration frequency (through the coordinated action of all three domains in a single molecular complex).
Solution Approach 2:
The polypeptide is designed as a universal multi-functional tool that can integrate various heterologous sequences at specific genomic locations. The DNA-binding domain can be adapted to recognize different target sequences, while the reverse transcriptase and endonuclease domains provide universal integration capability, making the system both specific and productive across different applications.
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 achieves precise and efficient insertion or deletion of nucleotides into target genomic sites, with capabilities ranging from 1 to 200 nucleotides, and can introduce heterologous sequences with high homology, addressing the limitations of existing technologies.
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
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.