Split Prime Editor Assembly for Efficient Nucleotide Editing
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Solution Overview
Problem
There is a need for split prime editors that facilitate prime editing with improved efficiency.
Innovation Solution
The development of split prime editors comprising a DNA binding domain and a DNA polymerase domain, where the domains are split into two polypeptides that passively assemble in a host cell to form the complete editor, utilizing affinity moieties, peptide tags, and intein sequences for assembly, and can include Cas9 proteins and reverse transcriptases for efficient nucleotide editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a single polypeptide is used to form the complete prime editor, then the editor structure is stable, but the delivery complexity and cellular assembly requirements increase
Solution Approach 1:
The prime editor is divided into two separate polypeptides: a first polypeptide comprising a DNA binding domain and a second polypeptide comprising a DNA polymerase domain. These separate polypeptides are delivered to the cell and passively assemble into the functional prime editor complex, simplifying delivery and assembly compared to a single complex polypeptide.
2Ease of operation
If the first and second polypeptides are delivered separately to the cell, then delivery flexibility is improved, but the assembly mechanism complexity increases
Solution Approach 1:
The first and second polypeptides passively assemble into the complete prime editor within the host cell without requiring external assembly machinery or complex mechanisms. The polypeptides self-assemble through their inherent affinity, enabling flexible separate delivery while maintaining simple assembly.
3Productivity
If affinity moieties are used to facilitate polypeptide assembly, then assembly efficiency is improved, but the protein sequence complexity increases
Solution Approach 1:
Affinity moieties act as intermediaries that facilitate the passive assembly of the first and second polypeptides into the complete prime editor. These affinity moieties enable efficient assembly by mediating the interaction between the separate polypeptides, improving productivity while adding manageable sequence complexity.
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
The split prime editors enhance the efficiency of prime editing by allowing passive assembly and integration into host cells, enabling precise nucleotide substitutions, insertions, and deletions, particularly in human cells, with potential applications in treating genetic diseases.
Implementation Method 1
the first polypeptide and the second polypeptide are configured to passively assemble in a host cell to form the split prime editor
Implementation Method 2
the first polypeptide has affinity for the second polypeptide. In some embodiments, the second polypeptide has affinity for the first polypeptide
Implementation Method 3
a first polypeptide comprising a first amino acid sequence, wherein the first amino acid sequence forms at least a portion of a DNA binding domain
Implementation Method 4
a second polypeptide comprising a second amino acid sequence, wherein the second amino acid sequence forms at least a portion of a DNA polymerase domain
Data Source
AI summary
Provided herein are compositions and methods related split prime editors.