GeoCas9 Nucleobase Editors for Broad PAM and Thermostable Targeting
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Solution Overview
Problem
Current genome engineering tools, such as spCas9, are large in size, require specific protospacer-adjacent motifs for binding, and have stability issues under varying temperature conditions, limiting their efficiency and precision in nucleic acid editing.
Innovation Solution
Development of GeoCas9 fusion proteins, comprising a Cas9 protein, a cytidine deaminase domain, and a uracil glycosylase inhibitor (UGI) domain, which can alter sequence specificity, bind to diverse PAM sequences, and operate at thermostable conditions, facilitating precise and efficient nucleic acid editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If spCas9 is used for nucleic acid editing, then DNA cleavage can be achieved, but the large size of spCas9 protein limits delivery efficiency
Solution Approach 1:
The patent extracts and removes specific domains from the spCas9 protein structure to create miniaturized Cas9 variants. By eliminating non-essential domains while preserving the core nuclease activity and PAM recognition capability, the protein size is reduced from approximately 1600 amino acids to smaller variants that can be more efficiently delivered while maintaining DNA cleavage function.
2Measurement precision
If spCas9 requires specific PAM sequences for binding, then target specificity is achieved, but the requirement for specific PAM sequences limits the range of editable sites
Solution Approach 1:
The patent employs parameter changes by mutating specific amino acid residues in the PAM recognition domain of Cas9. These mutations alter the biochemical parameters of PAM recognition, enabling the Cas9 variant to recognize different PAM sequences (such as NG, NGA, or other variations) while maintaining sufficient target specificity through the guide RNA-DNA hybridization mechanism.
3Productivity
If spCas9 is used under varying temperature conditions, then genome engineering can be performed, but stability issues reduce editing efficiency
Solution Approach 1:
The patent applies local quality changes by introducing stabilizing mutations specifically in the thermal stability-critical regions of the Cas9 protein structure. These localized modifications enhance the protein's thermostability and resistance to proteolytic degradation without compromising the catalytic activity or target binding capability, thereby improving editing efficiency under varying temperature conditions.
4Adaptability or versatility
If GeoCas9 fusion proteins are designed with modified PAM recognition, then versatility in target selection is improved, but sequence specificity may be reduced
Solution Approach 1:
The patent uses the guide RNA as an intermediary element that maintains high sequence specificity despite modifications in the Cas9 PAM recognition domain. The guide RNA-DNA hybridization provides a secondary layer of specificity that compensates for the reduced discrimination capability of the mutated Cas9 protein, ensuring that off-target effects are minimized while expanding PAM recognition range.
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
GeoCas9 fusion proteins enable precise and efficient nucleic acid editing by altering sequence specificity and stability, improving editing efficiency and reducing off-target activity.
Implementation Method 1
a cytidine deaminase domain
Implementation Method 2
GeoCas9 binds to its target sequence by denaturing the dsDNA, resulting in a stretch of DNA that is single-stranded
Data Source
AI summary
Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids or the modification of nucleic acids or proteins, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of nucleic acid programmable DNA binding proteins e.g., GeoCas9 or variants thereof, and effector domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing or protein modification are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a GeoCas9 and effector domains, are provided.