Fusion Polypeptides for Precise Genomic Editing
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Solution Overview
Problem
Current CRISPR/Cas systems face limitations in precisely introducing targeted modifications into the cell genome, particularly for modifying coding sequences associated with diseases, due to challenges in directing single-stranded DNA cleavage and genomic targeting.
Innovation Solution
Development of fusion polypeptides comprising a catalytically inactive Cpf1 domain and an endonuclease domain, such as FokI, that facilitate single-stranded DNA cleavage, combined with a genomic modification domain like a base editor, to enable precise genomic modifications by forming a ribonucleoprotein complex with guide RNA and binding to target sites in the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a catalytically inactive Cpf1 domain is used alone for genomic targeting, then the system maintains stability and avoids unwanted DNA cleavage, but it lacks the ability to perform single-stranded DNA cleavage for precise genomic modifications
Solution Approach 1:
The patent combines a catalytically inactive Cpf1 domain with an active endonuclease domain (such as FokI) to create a fusion polypeptide. This merging allows the system to maintain the stability and targeting precision of Cpf1 while gaining the single-stranded DNA cleavage capability of the endonuclease domain, resolving the contradiction between stability and functional capability.
2Manufacturing precision
If traditional CRISPR/Cas systems are used for genomic editing, then the systems can introduce double-stranded breaks, but they face limitations in precisely introducing targeted modifications due to challenges in directing single-stranded DNA cleavage
Solution Approach 1:
The patent segments the functional capabilities by using a catalytically inactive Cpf1 domain for precise targeting and guide RNA binding, while incorporating a separate active endonuclease domain for single-stranded DNA cleavage. This segmentation allows each component to specialize in its optimal function, improving precision while maintaining versatility.
Solution Approach 2:
The fusion polypeptide acts as a composite system combining elements with different functional properties: the Cpf1 domain provides RNA-guided DNA binding and PAM recognition, while the endonuclease domain provides cleavage activity. This composite structure enables precise single-stranded DNA cleavage that neither component could achieve alone.
3Productivity
If the endonuclease domain is designed to form dimers for DNA cleavage, then the system achieves enhanced cleavage efficiency, but it increases the complexity of the polypeptide structure
Solution Approach 1:
The patent merges two endonuclease domains within a single polypeptide chain to create a functional dimeric structure. This approach achieves the cleavage efficiency of dimeric endonucleases while simplifying the overall system architecture compared to using separate dimeric proteins, as the dimerization interface is built into the fusion construct itself.
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 approach allows for efficient and targeted introduction of mutations or modifications into the genome, overcoming previous limitations in precision and efficiency of CRISPR/Cas systems, enabling the generation of genetically engineered cells with specific edits.
Implementation Method 1
forming a ribonucleoprotein complex with guide RNA and binding to target sites in the genome
Implementation Method 2
function in directing single stranded DNA cleavage (i.e., nickase activity) to a target site in the genome of a cell
Data Source
AI summary
Provided herein are fusion polypeptides comprising a Cpf1 domain lacking nuclease activity and an endonuclease domain. Also provided herein are fusion polypeptides further comprising a genomic modification domain, which in some embodiments is a base editor, such as a deaminase. Also provided herein are methods involving contacting the fusion polypeptides with a gRNA to form a genetic editing system directed to a target site sequence in the genome of a cell.