Engineered CRISPR Nucleases for Specific Editing in Mature Cells
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
CRISPR/Cas systems face limitations in eukaryotic organisms, including inefficient delivery to mature cells, low editing efficiency, off-target events, and target sequence preferences, as well as suboptimal enzymatic activity conditions.
Innovation Solution
Engineered nucleases with specific amino acid sequences having varying degrees of identity and targeted substitutions to SEQ ID NOs: 1-465, 1063-1070, 1080-1081, and 1095, designed to enhance delivery, efficiency, and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas systems are used for nucleic acid editing, then editing capability is achieved, but delivery efficiency to mature cells is poor
Solution Approach 1:
The patent modifies the Cas9 nuclease by changing amino acid parameters at specific positions (K164R, N564R, N570R, Q827L substitutions) to create variants with improved properties. These parameter changes in the protein sequence enable better delivery to mature cells while maintaining editing capability, directly resolving the contradiction between reliability and productivity.
2Reliability
If standard CRISPR/Cas systems are used, then editing function is provided, but off-target events occur
Solution Approach 1:
The patent introduces specific amino acid substitutions (K164R, N564R, N570R, Q827L) in the Cas9 protein to modify its binding and cleavage properties. These parameter changes increase specificity for on-target sequences while reducing off-target effects, thereby resolving the contradiction between maintaining editing function and eliminating harmful off-target events.
3Reliability
If CRISPR/Cas nucleases are used, then nucleic acid editing is achieved, but target sequence preferences limit versatility
Solution Approach 1:
The engineered Cas9 variants with amino acid substitutions (K164R, N564R, N570R, Q827L) exhibit modified PAM recognition properties and reduced target sequence preferences compared to wild-type Cas9. These parameter changes in the nuclease structure enable broader target sequence compatibility while maintaining editing reliability, resolving the contradiction between reliability and versatility.
4Reliability
If conventional CRISPR/Cas systems are used, then editing is performed, but enzymatic activity conditions are suboptimal
Solution Approach 1:
The engineered Cas9 variants demonstrate improved enzymatic activity under a broader range of conditions including varied temperatures and buffer compositions. The amino acid substitutions enhance the stability and catalytic efficiency of the nuclease, making the system more robust and easier to operate across different experimental conditions while maintaining reliable editing performance.
Data Source
AI summary
The present disclosure provides nucleases and compositions, methods, and systems thereof for nucleic acid modification. More particularly, the present disclosure provides compositions and system comprising a nuclease comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-1096 and at least one gRNA.