Engineered Retrons for HDR Donor Template Delivery in Genome Editing
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Solution Overview
Problem
Existing methods for precise genome editing using programmable nucleases face inefficiencies in delivering donor DNA templates for homology-directed repair (HDR), limiting the effectiveness of genome editing processes.
Innovation Solution
Development of recombinant retrons with genetic modifications to enhance the production of msDNA donor templates, combined with programmable nucleases and guide RNAs, for efficient genome editing systems, including delivery via vectors and compositions such as plasmids, virus-based vectors, and lipid nanoparticles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If physical tethering of donor DNA to nuclease components is used, then delivery efficiency is improved, but HDR-dependent editing efficiency remains unsatisfactory
Solution Approach 1:
The patent introduces an RNA molecule as an intermediary carrier that physically tethers donor DNA to the CRISPR-Cas9 complex. This RNA intermediary contains both the guide sequence for Cas9 binding and the donor DNA template, enabling efficient delivery while maintaining high HDR-dependent editing efficiency by coordinating both functions in a single molecular structure
Solution Approach 2:
The patent combines the guide RNA and donor DNA template into a single fused RNA molecule structure. This merging eliminates the need for separate delivery components and ensures that the donor DNA is delivered precisely to the target site where the Cas9 complex is active, thereby improving both delivery efficiency and editing efficiency simultaneously
2Manufacturing precision
If donor DNA template is delivered to DSB site, then precise genome editing is achieved, but the process is limited by delivery efficiency
Solution Approach 1:
The RNA molecule is designed to self-assemble with the Cas9 protein and self-localize to the target DNA site through its guide sequence. The donor DNA template is inherently linked to the guide RNA structure, ensuring that the editing machinery automatically carries its own required template without external delivery assistance, thereby achieving both precision and high efficiency
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
Enhances the concentration and efficiency of donor DNA templates for HDR-dependent editing, improving the precision and effectiveness of genome editing in various cell types, including human and bacterial cells.
Implementation Method 1
The RT binds the RNA template downstream from the msd locus, initiating reverse transcription of the RNA towards its 5′ end
Implementation Method 2
The 2′OH group present in a conserved branching guanosine residue that acts as a primer
Implementation Method 3
the resulting DNA, the msDNA, remains covalently attached to the RNA template via a 2′-5′ phosphodiester bond
Implementation Method 4
The external regions, at the 5′ and 3′ ends of the msd/msr transcript (a1 and a2, respectively) are complementary and can hybridize
Data Source
AI summary
Disclosed are engineered retrons and methods of use such as to modify the genome of a host (e.g, mammalian) cell by delivering the engineered retron or the encoded ncRNA in vitro or in vivo to the host (e.g., mammalian) cell.