Evolved Base Editors Through PACE for Sequence-Context Agnostic Editing
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Solution Overview
Problem
Current base editors face limitations such as PAM requirements, off-target editing, production of undesired genomic byproducts, and low editing efficiencies, particularly in sequence-context-dependent manner, which hinder their effectiveness in targeted nucleic acid editing.
Innovation Solution
Development of evolved base editors using a phage-assisted continuous evolution (PACE) system to enhance editing efficiency and reduce sequence-context dependency, utilizing cytidine deaminases with specific mutations and fusion proteins comprising nucleic acid programmable DNA binding proteins and uracil glycosylase inhibitors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If base editors use naturally occurring Cas9 or modified versions with specific PAM requirements, then targeted editing can be achieved, but the utility is limited by PAM requirement constraints
Solution Approach 1:
The patent modifies the PAM recognition parameters by using engineered Cas9 variants with altered PAM specificities (e.g., xGA, NG, NAG, NxG motifs) instead of the canonical NGG requirement. This parameter change in PAM recognition expands the range of editable genomic sites while maintaining targeted editing capability through programmable guide RNA design.
2Productivity
If base editors are designed for high editing efficiency at specific sites, then editing efficiency improves, but off-target base editing of non-target nucleotides nearby the desired editing site occurs
Solution Approach 1:
The patent employs narrow editing window base editors that concentrate deaminase activity within a restricted 5-nucleotide window immediately adjacent to the PAM site. This local quality control ensures that only nucleotides within this precise window are edited, while nearby non-target nucleotides remain unaffected, thereby reducing off-target editing events.
Solution Approach 2:
The patent uses a programmable guide RNA as an intermediary that directs the base editor complex to the specific target sequence with high precision. The guide RNA mediates between the Cas9 moiety and the target DNA, ensuring that the deaminase activity is activated only at the intended editing site through proper R-loop formation, thereby preventing off-target editing.
3Adaptability or versatility
If base editors are used for targeted nucleic acid editing, then gene function study and disease therapy potential are enabled, but undesired edited genomic byproducts such as indels are produced
Solution Approach 1:
The patent extracts and removes the nuclease activity from the Cas9 moiety by using catalytically disabled Cas9 variants (dCas9 or nickase variants). This extraction of harmful nuclease function eliminates the production of indels and other undesired genomic byproducts, while preserving the programmable DNA binding capability needed for targeted base editing.
4Object-generated harmful factors
If base editors are designed with narrow editing windows to reduce off-target effects, then off-target editing is reduced, but the editing window is normally only about 5 nucleotides wide which limits targetability
Solution Approach 1:
The patent achieves universality by developing multiple base editor variants with different PAM specificities (xGA, NG, NAG, NxG) and different deaminase components (APOBEC1, CDA, AID). This multi-functionality allows the base editing system to target diverse genomic sequences with varying editing window requirements, making the technology universally applicable across different target sites while maintaining precise editing control.
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 evolved base editors demonstrate improved editing efficiency and sequence-context agnosticism, enabling more precise and effective targeted nucleic acid modifications, including therapeutic applications for genetic diseases.
Implementation Method 1
C-to-T editors use a cytidine deaminase to convert cytidine to uridine in the single-stranded DNA loop opened by Cas9
Implementation Method 2
The opposite strand is nicked by Cas9 to stimulate DNA repair mechanisms that use the edited strand as a template
Implementation Method 3
The base editor gene is cloned into a phage genome and expressed in a host cell. The host cell is then subjected to a selection pressure that favors the production of evolved base editors with improved editing efficiency
Data Source
AI summary
The instant specification provides for evolved base editors which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system. In particular, the instant specification provides for evolved cytidine base editors (e.g., based on APOBEC1, CDA, or AID cytidine deaminase domains) which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system.