Nucleic Acid-Guided Nucleases Genome Editing Specificity
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Solution Overview
Problem
Existing nucleases for genome editing face challenges such as difficulties in large-scale purification, delivery issues due to size, and limitations in specificity, processivity, genome editing efficiency, and targeting functionality due to PAM recognition sequences.
Innovation Solution
Development of novel nucleic acid-guided nucleases with unique characteristics that enhance target recognition specificity and genetic editing efficiency, including engineered nuclease systems with guide polynucleotides designed to hybridize with specific target sequences in eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing nucleases are used for genome editing, then genome editing functionality is achieved, but purification on large scale becomes difficult
Solution Approach 1:
The nuclease is divided into two separate components: a guide polynucleotide (crRNA or sgRNA) and a Cas protein. This segmentation allows the guide component to be easily synthesized and purified independently, while the Cas protein can be produced and purified separately, solving the purification difficulty while maintaining genome editing functionality.
Solution Approach 2:
The guide sequence is extracted as a separate functional element from the full-length CRISPR RNA, allowing it to be designed and synthesized independently. This extraction enables flexible combination with different Cas proteins and simplifies the purification process for both components.
2Productivity
If existing nucleases are used for genome editing, then genome editing functionality is achieved, but delivery becomes challenging due to size
Solution Approach 1:
The genome editing system is segmented into smaller components (guide polynucleotide and Cas protein) that can be delivered separately via different methods (e.g., RNA delivery, protein transduction, or separate plasmid transfection), overcoming the size limitations of delivering a single large nuclease complex.
Solution Approach 2:
The system allows dynamic assembly of the nuclease complex inside the target cell after separate delivery of components, providing flexibility in delivery timing and method while achieving the same genome editing outcome.
3Reliability
If existing nucleases are used for genome editing, then basic editing capability is provided, but specificity and targeting functionality are limited
Solution Approach 1:
The guide polynucleotide is designed with specific local sequences (20-nt spacer sequence) that provide high specificity for target recognition, while the Cas protein provides the catalytic function. This local quality differentiation enables high specificity without compromising versatility.
Solution Approach 2:
The Cas protein serves as a universal platform that can work with different guide polynucleotides targeting different genomic sequences. This multi-functionality allows a single Cas protein to provide versatile targeting capabilities across multiple genes and organisms while maintaining high specificity through guide sequence design.
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 new genome editing tools provide increased flexibility and improved genome editing efficiency and specificity, making them suitable for various applications including biomedical research, agriculture, and human gene therapy.
Implementation Method 1
the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell
Data Source
AI summary
The present disclosure provides novel nucleic acid-guided nucleases and methods of using the nucleases for genome editing. The present disclosure further provides a system for editing a target region in a genome comprising a nucleic acid-guided nuclease, a heterologous guide nucleic acid for complexing with the nucleic acid-guided nuclease, and an editing polynucleotide configured to bind to the target region.