Engineered CRISPR DNA Targeting Enzymes for Diverse Cells
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Solution Overview
Problem
Current CRISPR-Cas systems lack programmable effectors with unique PAM sequence requirements and are limited in their application to non-natural environments, such as bacteria other than those in which they were initially discovered or in eukaryotic cells like mammalian cells.
Innovation Solution
Development of engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems, including CRISPR-associated proteins with specific amino acid sequences and RNA guides, capable of binding to target nucleic acids and modifying them, adapted for use in diverse cellular environments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing CRISPR-Cas systems are used, then genome editing capability is provided, but the systems lack diversity in PAM sequence requirements and cannot be applied to diverse cellular environments
Solution Approach 1:
The effector protein is divided into two separate components: a nucleic acid-binding domain (such as a transcription factor domain) and a nuclease domain (such as Cas9 or Cas12a). These segmented components can function independently or in combination, allowing flexible adaptation to different cellular environments and target sequences without requiring a single complex multi-subunit protein assembly.
Solution Approach 2:
The system employs a universal nuclease domain (e.g., Cas9 or Cas12a) that can be paired with different nucleic acid-binding domains to create effectors with diverse specificities. This multi-functional approach allows the same core nuclease to target different PAM sequences and genomic locations by simply changing the binding domain, thereby achieving versatility across various cellular environments without increasing overall system complexity.
2Adaptability or versatility
If novel programmable effectors with unique PAM sequence recognition are developed, then application scope in non-natural environments is expanded, but the component set becomes more complex
Solution Approach 1:
By segmenting the effector into separate nucleic acid-binding domains and nuclease domains, the system allows independent optimization of each component. The nucleic acid-binding domain can be engineered for specific PAM recognition in non-natural environments, while the nuclease domain remains a standardized, well-characterized component. This segmentation simplifies the engineering process compared to designing entirely new multi-subunit effectors.
Solution Approach 2:
The system uses an intermediary nucleic acid-binding domain that mediates between the target sequence recognition and the nuclease activity. This intermediary component can be independently selected or engineered to provide the desired PAM specificity for non-natural environments, while the nuclease domain serves as a reliable effector module. This mediator approach reduces engineering complexity by separating the specificity-determining functions from the catalytic functions.
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
Enables efficient genome editing and modulation of gene expression in various cellular contexts, including mammalian cells, by providing novel programmable effectors with unique PAM sequence recognition and modification capabilities.
Implementation Method 1
an RNA guide including a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid
Implementation Method 2
the CRISPR-associated protein is capable of binding to the RNA guide and of modifying the target nucleic acid sequence complementary to the spacer sequence
Data Source
AI summary
The disclosure describes novel systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of nucleic acids. Each system includes one or more protein components and one or more nucleic acid components that together target nucleic acids.