Engineered Type V-I CRISPR-Cas Systems for Versatile Genome Editing
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Solution Overview
Problem
Current CRISPR-Cas systems have limitations in programmable effectors and systems for modifying nucleic acids beyond their capabilities, necessitating the development of additional technologies for genome and epigenome manipulation.
Innovation Solution
Engineered non-naturally occurring CRISPR-Cas systems, specifically Type V-I systems, with novel RNA guides and effector proteins that include RuvC domains, offering enhanced DNA/RNA editing properties, smaller size for versatile delivery, and genotype-triggered cellular processes such as cell death, along with programmable RNA-guided DNA insertion, excision, and mobilization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional CRISPR-Cas systems are used, then genome editing capability is provided, but the system lacks versatility for diverse nucleic acid modifications and has limited programmable effector options
Solution Approach 1:
The patent develops multiple Class 2 CRISPR-Cas systems (Cas9, Cas12a, Cas12b, Cas13) that can be programmed with different guide RNAs to target various nucleic acid sequences. Each effector protein provides different functional capabilities (DNA cutting, RNA cutting, base editing) while following a universal guide RNA-directed mechanism, enabling diverse nucleic acid modifications through a unified platform
Solution Approach 2:
The patent modifies effector proteins through targeted mutations to change their functional parameters. For example, Cas9 variants are engineered with altered PAM recognition specificities and different cutting behaviors (nickase vs. double-strand break). Guide RNA parameters are also optimized with different lengths and structures to match specific effector proteins, expanding the range of targetable sequences
2Reliability
If larger effector proteins are used, then more comprehensive DNA binding and cutting capability is achieved, but delivery versatility and ease of administration are reduced
Solution Approach 1:
The patent divides the CRISPR system into separate modular components: the effector protein (Cas9, Cas12a, etc.), the guide RNA, and the target DNA sequence. This segmentation allows each component to be independently optimized and delivered. Smaller effector proteins like Cas12a (1000 amino acids) and Cas13 (1300 amino acids) are selected to facilitate delivery while maintaining functional capability
Solution Approach 2:
The guide RNA serves as an intermediary that bridges the effector protein and target DNA. It contains the spacer sequence complementary to the target and structural elements that facilitate binding to the effector. This intermediary allows the effector to recognize and bind specific sequences without requiring the protein itself to be large or complex
3Adaptability or versatility
If existing CRISPR systems are used, then basic genome editing is achieved, but novel applications in epigenome manipulation and cellular control are limited
Solution Approach 1:
The patent combines CRISPR-Cas systems with additional functional domains to create multifunctional effectors. For example, dCas9 (catalytically dead Cas9) is fused with epigenetic modifiers, transcription factors, or other cellular control proteins. This merging allows the guide RNA-directed targeting capability of CRISPR to be combined with diverse cellular functions, enabling applications in epigenome manipulation, gene activation, and cellular reprogramming
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 new systems provide broad applications for specific, programmed perturbations in genome and epigenome manipulation, enabling novel applications beyond existing CRISPR-Cas systems by offering improved editing capabilities and cellular control.
Implementation Method 1
an RNA guide element that is responsible to targeting these protein activities to a specific sequence on the phage DNA or RNA. The crRNA consists of a direct repeat responsible for protein binding to the crRNA and a spacer sequence that is complementary to the desired nucleic acid target 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 such as DNA. Each system includes one or more protein components and one or more nucleic acid components that together target nucleic acids.