Engineered Cas9 Enzymes for Diverse PAM Recognition
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Solution Overview
Problem
Current CRISPR-Cas9 systems face challenges in achieving precise genomic modifications, particularly in requiring diverse tools for targeting various genomic sites and making single-base edits, which is not adequately addressed by existing genome editing technologies.
Innovation Solution
Identification and engineering of novel Cas9 enzymes from bacteria such as Streptococcus constellatus, Sharpea spp., Veillonella parvula, Ezakiella peruensis, Lactobacillus fermentum, and Peptoniphilus sp. Marseille-P3761, which recognize specific protospacer adjacent motifs (PAM) sequences, allowing for their expression in eukaryotic cells and targeting diverse genomic sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 systems are used for genome editing, then gene knockout or expression modification is achieved, but precise single-base editing and targeting of diverse genomic sites remain challenging
Solution Approach 1:
The patent applies universality by developing a platform that can perform multiple gene editing functions (knockout, precise base editing, insertion, deletion) using a unified Cas9 enzyme system. The engineered Cas9 enzymes recognize diverse PAM sequences, enabling the same system to target various genomic sites while maintaining precision through programmable guide RNAs.
Solution Approach 2:
The patent employs parameter changes by modifying the PAM recognition specificity of Cas9 enzymes through protein engineering. By changing the PAM sequence recognition parameters (from traditional NGG to alternative sequences), the system expands its targeting capability across diverse genomic sites while preserving editing precision through controlled enzymatic activity.
2Adaptability or versatility
If existing CRISPR tools are used, then basic genome editing is achieved, but diverse genomes and target sequences require expanded toolsets
Solution Approach 1:
The patent resolves this contradiction by creating a universal Cas9 platform that can handle diverse genome editing needs through a single system. The engineered Cas9 enzymes recognize multiple PAM sequence variants, eliminating the need for multiple specialized tools while maintaining the ability to target diverse genomic sequences across different genomes.
3Manufacturing precision
If precise genomic modifications are required, then single-base editing is needed, but current technologies lack adequate tools for this purpose
Solution Approach 1:
The patent applies parameter changes by precisely controlling the enzymatic activity parameters of Cas9 through protein engineering. By modifying amino acid residues in the Cas9 enzyme, the system achieves high-fidelity single-base editing while maintaining reliable targeting, balancing precision and success rate through optimized catalytic parameters.
Data Source
AI summary
The present invention provides novel systems, methods and compositions for making and using recombinantly engineered novel Cas9 enzymes optimized for human cells, for nucleic acid targeting and manipulation. The present invention is based on the discovery of novel Cas9 enzymes from Streptococcus constellatus, Sharpen spp. isolate RUG017, Veillonella parvula, Ezakiella peruensis, Lactobacillus fermentum strain AF15-40LB strain and Peptoniphilus sp. Marseille-P3761 bacteria that were codon-optimized and recombinantly produced for use in human cells. In some embodiments, novel Cas9 enzymes can be used for base editing. In some embodiments, the novel engineered Cas9 enzymes are used to treat human diseases.