Polycistronic mRNA Construct for Multiplex Genome Editing
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Solution Overview
Problem
Current gene editing tools, such as CRISPR/Cas9, base editors, and prime editors, have limitations in multiplexability and efficiency due to specific applications and bystander effects, requiring multiple tools for different editing tasks and lacking a single construct that can perform various editing functions efficiently.
Innovation Solution
Isolated nucleic acids encoding multiple genome editing enzymes like Cas, cytosine base editor, adenine base editor, prime editor, and regulatory elements, allowing for controlled expression of these enzymes using inducible promoters and destabilization domains, enabling flexible and efficient multiplex genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple separate gene editing tools are used for different editing tasks, then each specific editing function can be performed, but the device complexity and number of constructs required increases
Solution Approach 1:
The patent combines multiple gene editing tools (CRISPR/Cas9, base editors, prime editors) into a single polycistronic mRNA construct. This merging approach allows co-delivery of multiple editing enzymes through one construct, reducing the number of separate constructs needed while maintaining the ability to perform diverse editing functions including C-to-T conversions, A-to-G conversions, and small insertions/deletions
Solution Approach 2:
The single polycistronic mRNA construct is designed to encode multiple genome editing enzymes with different functions. By incorporating regulatory elements that enable independent control of each enzyme's expression, the construct achieves multi-functionality, allowing it to perform various editing tasks (disruption, base editing, prime editing) without requiring separate specialized constructs for each function
2Productivity
If multiple genome editing enzymes are expressed simultaneously, then multiplexed editing can be performed, but cross-talk between enzymes reduces editing precision
Solution Approach 1:
The patent employs dynamic, independent control of each genome editing enzyme's expression through specific regulatory elements (promoters, ribosome binding sites, polyadenylation signals) associated with each coding sequence in the polycistronic construct. This dynamic control allows the system to perform multiplexed editing while preventing cross-talk, as each enzyme can be independently regulated to achieve precise editing outcomes without interference from other enzymes
Solution Approach 2:
The polycistronic mRNA construct is segmented into distinct expression cassettes, each containing a coding sequence for a specific genome editing enzyme flanked by its own regulatory elements. This segmentation allows independent control of each enzyme's expression levels and timing, enabling multiplexed editing with high precision by preventing unwanted interactions between different editing enzymes
3Productivity
If a single construct encodes multiple editing enzymes, then delivery efficiency improves, but the size of the construct increases
Solution Approach 1:
The patent merges multiple coding sequences for different genome editing enzymes into a single polycistronic mRNA construct. This approach improves delivery efficiency by enabling co-delivery of multiple enzymes through one construct, reducing the total number of delivery events needed while the compact polycistronic design manages the construct size challenge
Data Source
AI summary
Provided herein are isolated nucleic acids comprising two or more nucleotide sequences capable of encoding genome-editing enzymes. Also provided are vectors comprising the isolated nucleic acids, host cells comprising the vectors, and kits comprising the isolated nucleic acids.