Fusion Proteins for Precision Editing via RDDP and Cas Segmentation
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Solution Overview
Problem
Current gene editing techniques, such as CRISPR, face challenges in achieving high specificity and efficiency for precision editing of a few nucleotides in the human genome, particularly in reducing off-target effects and improving editing efficiency.
Innovation Solution
The development of systems comprising an RNA-dependent DNA polymerase (RDDP), an effector protein (e.g., a CRISPR-associated Cas protein), a guide RNA, and a template RNA, which can be fused or linked as an extended guide RNA, to enhance precision editing by introducing specific nucleotide sequence modifications at target sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If commonly used reverse transcriptases (e.g., M-MLV) are used in precision editing systems, then the editing function can be achieved, but the enzyme size (around 650 amino acids) presents a challenge for insertion of coding sequences into the same delivery vector
Solution Approach 1:
The patent divides the precision editing system into separate functional components: a compact RDDP enzyme (less than 500 amino acids) and an effector protein (e.g., Cas protein), which can be delivered via separate vectors or as a fusion protein. This segmentation allows each component to be optimized independently for delivery and function.
Solution Approach 2:
The patent combines the RDDP and effector protein into a fusion protein to deliver both functions through a single vector. This merging simplifies the delivery system while maintaining both the compact enzyme size and editing functionality required for precision nucleotide editing.
2Ease of manufacture
If multiple vectors are used to deliver effector protein and reverse transcriptase separately, then the coding sequences can be inserted, but additional challenges arise for therapeutic use requiring multiple vectors to be formulated and delivered
Solution Approach 1:
The patent merges the RDDP and effector protein into a single fusion protein that can be delivered through one vector system, eliminating the need to formulate and deliver multiple separate vectors. This single-vector approach simplifies the delivery process while maintaining both enzymatic functions for precision editing.
3Manufacturing precision
If precision editing systems are used to introduce specific nucleotide edits, then editing specificity can be achieved, but editing efficiencies remain low
Solution Approach 1:
The patent optimizes the RDDP enzyme parameters by using a compact enzyme with less than 500 amino acids, which enhances editing efficiency while maintaining specificity. The enzyme's reduced size allows for improved kinetic properties and more efficient template switching during precision editing.
4Device complexity
If compact RDDPs with less than 500 amino acids are used, then coding sequences can be incorporated into a single delivery vector, but the enzyme must demonstrate enhanced editing efficiencies
Solution Approach 1:
The patent identifies and utilizes specific parameter changes in the RDDP enzyme structure (reducing to less than 500 amino acids) that simultaneously achieve compact size for vector compatibility and enhanced editing efficiency through improved enzymatic kinetics and template switching.
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
These systems demonstrate improved editing efficiencies and reduced off-target effects, enabling precise modifications of target genes and potential therapeutic applications.
Implementation Method 1
an RNA-dependent DNA polymerase (RDDP)... to introduce specific nucleotide sequence modifications at a target site
Implementation Method 2
the guide RNA and template RNA (rttRNA) are fused or linked as an extended guide RNA (e.g., rtgRNA)
Data Source
AI summary
Disclosed herein are composition and methods of modifying a target double-stranded DNA (dsDNA) molecule in a cell. Systems, compositions, and methods may comprise a CRISPR-associated (Cas) protein, an RNA-dependent DNA polymerase, and/or one or more guide nucleic acids or uses thereof.