DNA-Targeting RNA and Inactive Cas9 for Precise Gene Modulation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for genetic manipulation, such as RNA interference, face challenges with precision and off-target effects, requiring the design of new proteins for each target sequence and suffering from limited accuracy and toxicity.
Innovation Solution
A DNA-targeting RNA combined with a modifying polypeptide allows for site-specific modification of DNA, using an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA to modulate transcription or introduce specific enzymatic activities, such as nuclease activity, without the need for new protein design for each target sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If engineered nuclease enzymes are designed to target specific DNA sequences, then targeted gene deletion and replacement can be achieved, but each new genomic locus requires design of a novel nuclease enzyme making the approach time consuming and costly
Solution Approach 1:
The system divides the targeting function into two separate components: a reusable nuclease enzyme (Cas9) and a programmable RNA guide molecule. The RNA guide contains a variable spacer sequence that can be designed to match any target DNA sequence, while the Cas9 enzyme remains constant. This segmentation allows rapid adaptation to new targets by only changing the RNA guide rather than redesigning the entire nuclease enzyme.
Solution Approach 2:
The patent introduces an RNA guide molecule as an intermediary between the nuclease enzyme and the target DNA. This RNA guide acts as a programmable mediator that directs the Cas9 enzyme to specific genomic loci through base-pairing with complementary DNA sequences. The intermediary RNA allows the enzyme to be reused across multiple targets without modification.
2Manufacturing precision
If engineered nuclease enzymes are used for targeted gene manipulation, then site-specific DNA modification can be achieved, but limited precision can lead to unpredictable off-target effects
Solution Approach 1:
The system enhances local quality at the target site by requiring multiple matching conditions: the RNA guide must base-pair with the target DNA, and a specific protospacer adjacent motif (PAM) sequence must be present immediately adjacent to the target site. This multi-layered local recognition ensures high specificity and minimizes off-target effects by requiring precise local matches rather than relying solely on overall sequence similarity.
Solution Approach 2:
The system incorporates feedback mechanisms through the PAM requirement and RNA-DNA hybridization stability checks. The Cas9 enzyme only proceeds with cleavage when both the RNA guide properly hybridizes to the target DNA and the correct PAM sequence is recognized. This feedback control ensures that nuclease activity is activated only at genuine target sites with appropriate molecular signatures, reducing erroneous off-target cleavage events.
3Adaptability or versatility
If RNA interference is used to target arbitrary genes for regulation, then gene expression can be modulated, but significant off-target effects and toxicity are exhibited
Solution Approach 1:
The patent replaces the RNAi mechanism (which uses RNA to recruit Dicer and RISC complexes for mRNA degradation) with a DNA-directed mechanism using CRISPR RNA guides and Cas nucleases. This substitution changes the molecular machinery from an RNA-centric system with inherent off-target binding issues to a DNA-centric system where specificity is determined by RNA-DNA hybridization and PAM recognition, fundamentally reducing off-target effects and cellular toxicity.
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
This approach enables precise and controlled site-specific modification of DNA with reduced off-target effects, facilitating targeted gene editing and expression modulation while minimizing the need for novel protein design.
Implementation Method 1
a targeting sequence, wherein the targeting sequence forms a hybridization duplex with a complementary sequence in the target DNA or RNA
Implementation Method 2
contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA
Implementation Method 3
together with a modifying polypeptide, provides for site-specific modification of a target DNA
Data Source
AI summary
The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.