Hybrid DNA/RNA CRISPR Guides for Specific Genome Cleavage
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Solution Overview
Problem
Existing CRISPR/Cas systems face issues with RNA-DNA hybrid structures, such as RNase A degradation and higher RNA-DNA mismatches, leading to inefficiencies and increased costs in RNA oligonucleotide synthesis, and lack specificity in target recognition.
Innovation Solution
A hybrid DNA/RNA polynucleotide system is introduced for Class 2 CRISPR systems, combining a targeting region of DNA with an activating region of RNA, which can be upstream or downstream, and interacting with Cas9 or Cpf1 proteins, forming structures like stem loops to enhance specificity and reduce off-target modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNA-based CRISPR guide sequences are used, then the system can naturally hybridize with target DNA, but the RNA strand is susceptible to RNase A degradation and forms unstable RNA-DNA hybrids
Solution Approach 1:
The patent changes the chemical composition parameter of the guide sequence from RNA to DNA, creating a DNA-based CRISPR guide that is resistant to RNase A degradation while maintaining hybridization capability with target DNA sequences
Solution Approach 2:
The patent creates a hybrid DNA-RNA system where the guide sequence is DNA and the target is DNA, forming a stable DNA-DNA hybrid that combines the stability of DNA with the sequence-specific recognition capabilities of the CRISPR system
2Ease of manufacture
If RNA guide sequences are used, then the CRISPR system can be implemented, but synthesis costs increase and robustness decreases
Solution Approach 1:
The patent replaces expensive and fragile RNA oligonucleotides with cheaper and more robust DNA oligonucleotides as the guide sequence, reducing synthesis costs and improving handling robustness while maintaining functional equivalence
3Measurement precision
If RNA-based CRISPR systems are used, then target recognition can occur, but specificity is reduced due to higher RNA-DNA mismatch tolerance
Solution Approach 1:
The patent changes the chemical nature of the guide sequence from RNA to DNA, which alters the hybridization properties and reduces tolerance for mismatches, thereby improving target recognition specificity and reducing off-target effects
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
The hybrid DNA/RNA polynucleotide system improves target specificity and reduces off-target effects, offering cost-effective and robust genome editing by preferentially cleaving at target sequences.
Implementation Method 1
a targeting region comprising deoxyribonucleic acid (DNA) and configured to hybridize with a target sequence in a nucleic acid
Implementation Method 2
wherein the polynucleotide forms a complex with a site-directed polypeptide and wherein the target nucleic acid is cleaved
Data Source
AI summary
The present disclosure provides DNA-guided CRISPR systems; polynucleotides comprising DNA, RNA and mixtures thereof for use with CRISPR systems; and methods of use involving such polynucleotides and DNA-guided CRISPR systems.