Engineered Nucleic Acid-Guided Nucleases for Precise Genome Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR/Cas9 and Cas12a systems face limitations in efficiency and accuracy for targeted genome editing, particularly in commercial applications, with issues such as imprecise editing and off-target effects.
Innovation Solution
Development of engineered nucleic acid-guided nucleases with altered recognition and improved PAM specificity, using engineered Cas12a-like nucleases with modified gRNAs and novel PAM sequences, including split gRNAs, to enhance targeting and reduce off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas9 or Cas12a systems are used for genome editing, then genome editing capability is achieved, but editing precision and accuracy are compromised due to off-target effects and imprecise editing
Solution Approach 1:
The patent modifies key parameters of the CRISPR/Cas system including PAM sequence recognition, guide RNA structure, and nuclease activity to achieve higher editing precision. Specifically, it engineers nucleases with altered PAM specificities and modified guide RNA sequences to reduce off-target effects while maintaining on-target editing efficiency.
Solution Approach 2:
The patent creates engineered variants of existing nucleases (Cas9, Cas12a) by copying and modifying their protein sequences, guide RNA structures, and associated components. These engineered copies exhibit improved specificity and reduced off-target activity compared to the wild-type systems.
2Measurement precision
If engineered nucleases with altered recognition are developed, then editing accuracy is improved, but system complexity increases
Solution Approach 1:
The patent segments the CRISPR/Cas system into modular components including separate guide RNA molecules, engineered nuclease proteins, and optional donor DNA cassettes. This segmentation allows independent optimization of each component's specificity and function while maintaining overall system manageability.
Solution Approach 2:
The patent introduces dynamic elements such as chemically modified guide RNAs with adjustable stability, temperature-sensitive nuclease variants, and inducible expression systems that can be activated or deactivated as needed. These dynamic features enable fine-tuning of editing precision without permanent system complexity.
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 engineered nucleases demonstrate increased efficiency and accuracy in genome editing, enabling precise and reliable editing in human and other species, including reduced off-target activity and improved editing capabilities.
Implementation Method 1
an engineered nucleic acid-guided nuclease comprising an engineered nuclease polypeptide and a guide nucleic acid
Implementation Method 2
Cas12a is capable of cleaving DNA distal to its PAM around the +18/+23 position of the protospacer. This cleavage creates a staggered DNA overhang (e.g. sticky ends)
Data Source
AI summary
Embodiments disclosed herein include novel nucleic acid-guided nucleases, novel guide nucleic acids, and novel targetable nuclease systems, and methods of use. In some embodiments, engineered non-naturally occurring nucleic acid-guided nucleases, can be used with known guide nucleic acids in a targetable nuclease system. In certain embodiments, targetable nuclease systems can be used to edit targeted genomes of humans and other species. In some embodiments, methods include, but are not limited to, recursive genetic engineering and trackable genetic engineering methods.