Compact RNA-Guided Gene Editing System
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current gene editing systems face challenges in efficiently targeting specific DNA sequences due to the large size of Cas9 enzymes and sequence specificity requirements, while smaller enzyme components like Zinc finger nucleases and TALENs are difficult to engineer for specific DNA binding.
Innovation Solution
A non-naturally occurring gene editing system comprising a targeting RNA, a guide RNA, and a polypeptide with a guide RNA-binding domain and a cleavage domain, where the polypeptide includes fewer than approximately 1200 amino acids, allowing for easy engineering and compact gene editing without sequence specificity requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Cas9 nuclease-based systems are used for gene editing, then the system can be directed to cleave specific DNA sequences by hybridizing targeting RNA to the target site, but the large size of the Cas9 enzyme makes it nearly impossible to incorporate the system into a single vector for gene delivery in humans
Solution Approach 1:
The invention divides the gene editing system into separate functional components: a compact polypeptide (guide RNA-binding domain + cleavage domain) and separate RNA molecules (guide RNA and targeting RNA). This segmentation allows the enzyme component to be small enough for vector delivery while maintaining specific DNA targeting through the RNA molecules.
Solution Approach 2:
The invention extracts and removes the large Cas9 enzyme from the system, retaining only the essential functional domains (guide RNA-binding domain and cleavage domain) in a compact polypeptide. The DNA sequence recognition function is extracted and assigned to separate RNA molecules, enabling the enzyme to be small while preserving targeting capability.
2Measurement precision
If Cas9 nuclease-based systems are used for gene editing, then the system can target specific DNA sequences, but the sequence requirements of the Cas9 enzyme itself (e.g., PAM sequence) limit the sites to which it can be targeted
Solution Approach 1:
The invention creates a universal gene editing platform where the compact polypeptide can work with different guide RNA sequences to target various DNA sites. The separation of targeting function (in RNA) from catalytic function (in polypeptide) makes the system adaptable to different target sequences without being constrained by fixed sequence requirements like PAM sequences.
Solution Approach 2:
The invention changes the targeting mechanism from protein-DNA direct recognition (Cas9 with PAM requirements) to RNA-DNA hybridization (guide RNA with targeting RNA). This parameter change in the recognition mechanism removes sequence specificity constraints, allowing the system to target a broader range of DNA sites by simply changing the guide RNA sequence.
3Weight of moving object
If Zinc finger nuclease and TALEN-based gene editing systems are used, then the enzyme components are much smaller, but the systems are hampered by the use of protein-based DNA-targeting domains, which are difficult to engineer to bind specific DNA target sequences
Solution Approach 1:
The invention replaces the mechanical/protein-based DNA recognition system (protein domains directly binding DNA) with an RNA-based recognition system. RNA molecules can base-pair with DNA targets through complementary base pairing, which is much easier to engineer and design than protein-DNA interactions, while keeping the enzyme component small.
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 and specific gene editing by using a compact enzyme system guided by easy-to-engineer RNA components, overcoming the limitations of existing technologies in terms of size and specificity.
Implementation Method 1
a first targeting RNA capable of hybridizing with a target DNA sequence
Implementation Method 2
a first guide RNA capable of binding to a first guide RNA-binding domain
Implementation Method 3
a polypeptide including the first guide RNA-binding domain and a first cleavage domain
Data Source
AI summary
The present invention provides RNA-guided gene editing systems and methods of use thereof.