Retrotransposon Genome Editing for Large Nucleic Acid Insertion
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Solution Overview
Problem
Current genome editing systems, such as CRISPR/Cas9, TALEN, and zinc finger proteins, are limited by size, cargo capacity, and targeting ability, particularly as they cannot efficiently insert large nucleic acids into specific genomic loci other than predetermined sites like the 28S rRNA locus.
Innovation Solution
A genome editing system comprising an R2 element enzyme with a reverse transcriptase and nickase domain, combined with a payload RNA that includes an insertion template and optional homology regions, allows for targeted insertion of large nucleic acids into specific genomic loci using a mechanism independent of endogenous cellular repair pathways.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If CRISPR/Cas9, TALEN, or zinc finger proteins are used for genome editing, then genome editing capability is achieved, but cargo capacity and ability to insert large nucleic acids is limited
Solution Approach 1:
The patent merges the R2 retrotransposon enzyme system with payload RNA containing insertion templates, combining the advantages of retrotransposon-based large cargo delivery with programmable targeting capabilities. This fusion allows the system to carry and insert large nucleic acid sequences that exceed the cargo capacity of traditional CRISPR/Cas9, TALEN, or zinc finger systems.
Solution Approach 2:
The payload RNA acts as an intermediary carrier that bridges the R2 enzyme system and the genomic target site. The RNA intermediate enables the transfer of large nucleic acid sequences from the enzyme complex to the genome, solving the cargo capacity limitation while maintaining insertion efficiency through the natural retrotransposon mechanism.
2Adaptability or versatility
If R2 element is used for insertion, then large nucleic acid cargo capacity is achieved, but targeting ability to specific loci is limited to predetermined sites like 28S rRNA locus
Solution Approach 1:
The patent introduces dynamic targeting capability by allowing the R2 enzyme system to be programmed with different guide RNAs that direct insertion to various genomic loci. This transforms the static, predetermined targeting of natural R2 elements into a dynamic, programmable system that can adapt to different target sites while maintaining precision through guide RNA-directed recognition.
Solution Approach 2:
The modified R2 enzyme system achieves universality by combining the large cargo capacity of retrotransposons with the programmable targeting of guide RNAs. This multi-functional system can target diverse genomic loci and insert various types of large nucleic acid payloads, making it versatile for different genome editing applications beyond the limited 28S rRNA locus.
3Reliability
If traditional genome editing systems are used, then editing capability is achieved, but size and complexity of the system increases
Solution Approach 1:
The patent extracts and utilizes only the essential components needed for large cargo insertion from the retrotransposon system (the R2 enzyme and payload RNA mechanism), omitting unnecessary elements of traditional genome editing systems. This streamlined approach maintains editing reliability for large nucleic acid insertion while reducing overall system complexity compared to using full CRISPR/Cas9, TALEN, or zinc finger 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
Enables directed insertion of large nucleic acids into specific genomic loci, including those other than the 28S rRNA locus, with enhanced targeting and cargo capacity, facilitating precise genome editing and therapeutic applications.
Implementation Method 1
Retrotransposons are mobile elements that insert themselves into the genome of a host through an RNA intermediate. The R2 element contains a single open reading frame (ORF), which encodes a reverse transcriptase, an endonuclease... The R2 element inserts itself into a host genome through a mechanism known as Target Primed Reverse Transcription (TPRT), which is a stepwise reaction including a first nick of host DNA, reverse transcription of the R2 RNA into the first strand
Implementation Method 2
Site-specific non-LTR retrotransposons are generally characterized by the presence of specific activity—reverse transcriptase activity, DNA nicking activity, and nucleic acid binding activity. The R2 element inserts itself into a host genome through a mechanism known as Target Primed Reverse Transcription (TPRT), which is a stepwise reaction including a first nick of host DNA, reverse transcription of the R2 RNA into the first strand, a second nick of host DNA
Data Source
AI summary
Genome editing tools for use in systems designed to deliver large genetic elements are disclosed herein. A genome editing system is described, which includes i) an R2 element enzyme or other non-LTR site specific retrotransposon element and ii) a payload RNA, wherein the payload RNA comprises an insertion region and optionally one or more of a 5′ homology region, a 3′ homology region, and a protein binding element, wherein the insertion region comprises a template for a small or large nucleic acid insertion into the genome, and wherein the R2 element enzyme or other non-LTR site specific retrotransposon element comprises a targeting domain, a reverse transcriptase domain, and a nickase domain. Also disclosed are cells edited using such a genome editing system, methods for editing a genome, and compositions comprising cells edited with this genomic editing system.