Genome Modulation with Retrotransposase-Mediated Long-Sequence Insertion
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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 require multiple steps or specialized proteins.
Innovation Solution
A system comprising a polypeptide with a reverse transcriptase and endonuclease domain, combined with a template RNA or DNA, enables precise and efficient insertion of heterologous sequences into a genome without the need for DNA-dependent polymerization or homologous recombination, using avian retrotransposase-derived components.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR/Cas9 is used for genome integration, then site specificity is improved, but effectiveness for integrating longer sequences deteriorates
Solution Approach 1:
The invention divides the integration function into separate components: a specialized protein for promoting insertion events and a template for providing the sequence to be integrated. This segmentation allows each component to be optimized independently, with the specialized protein handling the insertion mechanism and the template providing the genetic sequence, thereby enabling effective integration of longer sequences while maintaining site specificity.
Solution Approach 2:
The invention introduces a specialized protein as an intermediary that facilitates the integration process. This mediator protein promotes the insertion of the template sequence into the genome at the desired location, bridging the gap between the template and the host genome. This intermediary approach enables effective integration of longer sequences without compromising site specificity, as the specialized protein guides the template to the precise location.
2Productivity
If Cre/loxP approach is used for genome integration, then integration capability is improved, but process complexity deteriorates due to requiring multiple steps
Solution Approach 1:
The invention merges the functions of the Cre/loxP system into a single integrated approach. Instead of requiring separate steps for inserting loxP sites and then inserting the sequence of interest, the invention combines these functions into one process where a specialized protein directly promotes the insertion of the template sequence containing the heterologous object sequence into the genome at the desired location.
Solution Approach 2:
The invention performs preliminary action by incorporating the heterologous object sequence directly into the template that is introduced into the cell. This template already contains the complete sequence information and regulatory elements needed for integration, eliminating the need for subsequent steps to insert additional sequences or sites. The template is prepared in advance with all necessary components for direct integration.
3Device complexity
If existing integration methods are used, then simplicity of approach is improved, but insertion frequency and site specificity deteriorate
Solution Approach 1:
The invention employs a specialized protein that possesses inherent ability to promote insertion events without requiring additional external factors or complex machinery. The template RNA or DNA encoding the heterologous object sequence serves itself by being directly introduced into the genome at the desired location with the help of the specialized protein, which facilitates the insertion process naturally, thereby increasing insertion frequency while maintaining simplicity.
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 system allows for the targeted and efficient integration of exogenous genetic elements into a genome, achieving high specificity and insertion of sequences up to 7,500 amino acids without causing double-strand breaks or activating DNA repair pathways.
Implementation Method 1
a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain
Implementation Method 2
a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) an endonuclease domain
Data Source
AI summary
Methods and compositions for modulating a target genome are disclosed. The composition may comprise a first RNA encoding a polypeptide comprising a retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain. The composition may also comprise a second RNA comprising a sequence that binds the polypeptide and a heterologous object sequence. The composition may insert the sequence of the heterologous object sequence into a target DNA.