Engineered Cas9 Nuclease PAM Specificity for Genome Editing
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Solution Overview
Problem
Current CRISPR-Cas systems for genome editing face limitations in efficiency and accuracy, particularly in mammalian genomes, with challenges in precise editing and high off-target rates, which hinder their application in commercial and therapeutic contexts.
Innovation Solution
Development of novel nucleic acid-guided nucleases and guide nucleic acids, including engineered nucleases and gRNAs, that target specific genomes with improved specificity and reduced off-targeting, utilizing sequences such as those represented by SEQ ID NO: 94, 29, 81, 107, and gRNAs like those represented by SEQ ID NO: 125, 120, 124, 126, 118, 119, 121, 122, 123, 127, and 128, to enhance genome editing precision and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas systems are used for genome editing, then genome editing capability is achieved, but off-target effects increase and editing accuracy decreases
Solution Approach 1:
The patent applies parameter changes by modifying the PAM recognition specificity of Cas9 nucleases through mutagenesis. Specific amino acid substitutions in the PAM-interacting domain alter the PAM sequence requirements, enabling discrimination between on-target and off-target sites. This changes the biochemical parameters of nuclease recognition to improve editing accuracy while maintaining genome editing capability.
Solution Approach 2:
The patent applies local quality by introducing specific mutations only in the PAM-interacting domain of the Cas9 protein, while preserving the rest of the nuclease structure and function. This localized modification allows the system to maintain high editing capability at the target site while gaining improved specificity through altered PAM recognition at the binding interface.
2Productivity
If CRISPR-Cas systems are used for genome editing, then genome editing capability is achieved, but off-target rates increase
Solution Approach 1:
The patent changes the recognition parameters of the Cas9 nuclease by mutating amino acids in the PAM-interacting domain. This alters the PAM sequence specificity, allowing the system to distinguish between legitimate target sites and potential off-target sites, thereby reducing off-target effects while preserving on-target editing capability.
Solution Approach 2:
The patent converts the potential harm of broad Cas9 recognition specificity (which causes off-target effects) into a benefit by engineering restricted PAM specificity. The mutated Cas9 variants recognize only specific PAM sequences, transforming the general recognition capability into a selective recognition system that reduces off-target activity.
3Manufacturing precision
If novel engineered nucleases are developed, then editing accuracy improves, but system complexity increases
Solution Approach 1:
The patent achieves improved editing accuracy by changing the amino acid sequence parameters of existing Cas9 nucleases through targeted mutagenesis. This creates engineered variants with modified PAM specificity without requiring entirely new nuclease systems, thereby improving accuracy while minimizing the increase in system complexity.
Solution Approach 2:
The patent maintains universality by using the same Cas9 nuclease platform for multiple applications. The engineered variants with different PAM specificities can all function within the existing CRISPR-Cas9 system framework, allowing a single platform to serve multiple editing needs with varying specificity requirements.
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
These novel systems demonstrate increased enzymatic activity and editing accuracy, offering improved genome editing capabilities with reduced off-target effects, thereby facilitating more precise and efficient modifications in mammalian genomes.
Implementation Method 1
a guide polynucleotide (e.g. a guide RNA (gRNA)) that directs by homology the nuclease to cut a specific region of the genome
Implementation Method 2
the nuclease to cut a specific region of the genome
Data Source
AI summary
Embodiments disclosed herein include novel nucleic acid-guided nucleases, novel guide nucleic acids, and novel targetable nuclease systems, and methods of use. In some embodiments, engineered non-naturally occurring nucleic acid-guided nucleases, can be used with known guide nucleic acids in a targetable nuclease system. In certain embodiments, targetable nuclease systems can be used to edit targeted genomes of humans and other species. In some embodiments, methods include, but are not limited to, recursive genetic engineering and trackable genetic engineering methods.