HEPN Domain Endonucleases for Precise, PAM-Flexible Gene Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR-Cas systems face limitations such as off-target activities and protospacer adjacent motif (PAM) specificities, which restrict their use in gene editing and delivery.
Innovation Solution
Development of endonucleases with higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains, or fragments and variants thereof, with specific identities and amino acid modifications, to enhance target specificity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current CRISPR-Cas9 systems are used for gene editing, then high-efficiency gene modifications can be achieved, but off-target activities occur causing undesirable modifications at non-target sites
Solution Approach 1:
The patent applies parameter changes by modifying the biochemical properties of the endonuclease through HEPN domain engineering. Specific amino acid substitutions in the HEPN domain alter the enzyme's catalytic activity and target recognition parameters, enabling reduced off-target effects while maintaining on-target efficiency. This involves changing the chemical characteristics of the endonuclease active site to improve specificity.
2Adaptability or versatility
If current endonucleases are used, then gene editing can be performed, but use is restricted due to protospacer adjacent motif (PAM) specificities
Solution Approach 1:
The patent changes the PAM recognition parameters by engineering the HEPN domain to recognize different or relaxed PAM sequences. This allows the endonuclease to target a broader range of genomic sites that were previously inaccessible due to strict PAM requirements, thereby increasing adaptability without significantly increasing system complexity.
3Productivity
If current endonucleases are used, then gene editing can be performed, but packaging constraints limit delivery of system components
Solution Approach 1:
The patent extracts and optimizes the essential HEPN domain from the full CRISPR-Cas9 system, creating a minimized endonuclease variant that retains gene editing functionality while reducing overall protein size. This extracted HEPN domain can be more efficiently packaged and delivered to target cells, overcoming packaging constraints while maintaining productivity.
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 HEPN domain-containing endonucleases provide improved target specificity and reduced off-target effects, enabling more precise gene editing and delivery in various organisms.
Implementation Method 1
CRISPR-Cas systems thereby confer adaptive immunity in bacteria and archaea via RNA-guided nucleic acid interference
Implementation Method 2
The HEPN domain-containing endonucleases provide improved target specificity and reduced off-target effects, enabling more precise gene editing and delivery
Data Source
AI summary
The disclosure relates to compositions and methods that modify target nucleic acids, as well as methods of detecting nucleic acids. Various compositions are described herein, including compositions comprising endonucleases, endonuclease systems, and chimeric proteins having the endonuclease and a nucleic-acid modulating domain or a nucleic acid modifying domain.