Nme2Cas9-Deaminase Base Editing for Broader PAM Targeting
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Solution Overview
Problem
Conventional SpyCas9 base editors have limited editing windows and high off-target effects, making them inadequate for accurately targeting single-base mutations in human diseases.
Innovation Solution
The use of an NmeCas9 nuclease fused with a nucleotide deaminase protein, such as Nme2Cas9, which includes a nuclear localization signal and a protospacer accessory motif, allows for precise single nucleotide base editing by recognizing a diverse range of PAM sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If SpyCas9 base editor platforms are used, then base editing can be performed, but the editing window is limited and off-target effects are high
Solution Approach 1:
The patent changes the PAM recognition parameter from SpyCas9's NGG requirement to Nme2Cas9's N4CC requirement, fundamentally expanding the range of targetable sites. This parameter change in PAM specificity allows the editing system to access previously unreachable genomic locations while maintaining editing precision through the deaminase fusion mechanism.
Solution Approach 2:
The Nme2Cas9-deaminase fusion protein serves multiple functions: it provides PAM recognition (N4CC), enables base editing through deaminase activity, and achieves both high precision and broad applicability. This multi-functional design resolves the contradiction by making a single platform that is both precise and versatile across different genomic contexts.
2Reliability
If SpyCas9 base editor platforms are used, then base editing can be performed, but off-target effects are high
Solution Approach 1:
The deaminase enzyme acts as an intermediary that enables precise base conversion without requiring DNA double-strand breaks. This intermediary mechanism replaces the harmful nuclease activity with a safer chemical conversion process, reducing off-target effects while maintaining editing accuracy through the guided deaminase fusion protein.
3Adaptability or versatility
If Nme2Cas9-deaminase fusion proteins are used, then editing window is expanded and off-target effects are reduced, but protein complexity increases
Solution Approach 1:
The patent merges the Nme2Cas9 nuclease with the deaminase enzyme into a single fusion protein. This combining of components achieves multiple functions (PAM recognition, DNA binding, and base editing) in one molecular entity, expanding the editing window while managing complexity through functional integration rather than separate molecular components.
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 provides a highly accurate and precise gene editing platform with an expanded editing window, reducing off-target effects and enabling effective correction of single-base mutations associated with genetic disorders.
Implementation Method 1
such single nucleotide base editing results in a conversion of a C⋅G base pair to a T⋅A base pair
Data Source
AI summary
The present invention is related to the field of gene editing. In particular, the gene editing is directed toward single nucleotide base editing. For example, such single nucleotide base editing results in a conversion of a OG base pair to a T*A base pair. The high accuracy and precision of the presently disclosed single nucleotide base gene editor is accomplished by an NmeCas9 nuclease that is fused to a nucleotide deaminase protein. The compact nature of the NmeCas9 coupled with a larger number of compatible protospacer adjacent motifs provide the Cas9 fusion constructs contemplated herein to have a gene editing window that can edit sites that are not targetable by other conventional SpyCas9 base editor platforms.