C2c2 RNA-Targeting CRISPR System for Scalable Genome Editing
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Solution Overview
Problem
Current genome and transcriptome engineering technologies lack affordable, easy-to-set-up, scalable, and versatile methods for targeting multiple positions within eukaryotic genomes and transcriptomes, limiting their applications in genome engineering and biotechnology.
Innovation Solution
The development of Class 2 type VI CRISPR-Cas effector protein C2c2, which is a RNA-guided RNase capable of programmed RNA cleavage, allowing for specific targeting and modification of nucleic acid sequences, including the introduction of strand breaks, and can be used for various applications such as gene editing and cell regulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional genome-editing techniques (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbations can be achieved, but the methods are complex, expensive, and difficult to scale for multiple positions
Solution Approach 1:
The patent uses CRISPR-Cas9 systems where a guide RNA (copy of the target sequence) directs the Cas9 enzyme to the genomic target, replacing complex protein-DNA recognition systems with a simpler RNA-mediated copying mechanism that maintains precision while reducing complexity
Solution Approach 2:
The CRISPR-Cas9 system provides a universal platform that can target multiple genomic positions by simply changing the guide RNA sequence, whereas traditional methods require designing entirely new protein systems for each target, enabling scalable multi-position editing
2Reliability
If traditional genome-editing techniques are used, then genome perturbation is possible, but the methods are not affordable and difficult to set up
Solution Approach 1:
The patent replaces complex mechanical protein-DNA binding systems with a biochemical RNA-DNA hybridization system, which follows natural base-pairing rules and can be delivered via simple transfection methods, dramatically improving ease of setup while maintaining reliability
Solution Approach 2:
The guide RNA automatically finds and binds its complementary target sequence through natural hybridization, eliminating the need for complex delivery mechanisms or activation steps required by traditional methods, making the system both reliable and easy to implement
3Measurement precision
If traditional genome-editing techniques are used, then targeted perturbation can be achieved, but scaling to multiple positions is difficult
Solution Approach 1:
The patent segments the targeting function into two independent components: the Cas9 enzyme (effector) and the guide RNA (address). This allows multiple guide RNAs to be combined with a single Cas9 system, enabling simultaneous targeting of multiple positions while maintaining individual targeting accuracy through each guide's specific sequence
Solution Approach 2:
The patent merges multiple guide RNAs into a single CRISPR array transcript that is processed into individual guides, or combines multiple plasmid constructs encoding different guides with one Cas9 expression construct, enabling multiplexed editing that maintains the precision of single-target systems
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
C2c2 provides a robust and versatile tool for precise RNA targeting and modification, enabling efficient genome editing and transcriptome perturbation, with potential applications in biotechnology and medicine, including cancer treatment and disease modeling.
Implementation Method 1
C2c2 can be programmed to cleave ssRNA targets carrying protospacers flanked by a 3′H (non-G) PAM
Implementation Method 2
Cleavage is mediated by catalytic residues in the two conserved HEPN domains of C2c2
Data Source
AI summary
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.