Target-Specific SpCas9 Engineering to Reduce Off-Target Editing
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Solution Overview
Problem
The existing CRISPR/Cas9 system suffers from low target specificity, leading to unintended modification of non-target genes or nucleic acids due to partial complementary binding, which hampers efficient genome and epigenome manipulation.
Innovation Solution
An artificially engineered CRISPR enzyme, specifically a SpCas9 variant, is developed with improved target specificity by manipulating the end-capping loop and other regions of the SpCas9 protein, including amino acid deletions and substitutions, to enhance its ability to bind specifically to target sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the CRISPR-Cas9 system is used to modify target genes, then genome editing efficiency is improved, but off-target modification of non-target genes occurs due to partial complementary binding
Solution Approach 1:
The patent applies local quality by making specific amino acid substitutions at particular positions (e.g., Q979L, N984Y, T1000I) within the SpCas9 protein structure. These localized modifications in the PAM-interacting domain and end-capping loop region enhance discrimination between target and non-target sequences without compromising overall genome editing efficiency.
Solution Approach 2:
The patent changes the biochemical parameters of the Cas9 enzyme by substituting amino acids with different properties (e.g., changing charge, hydrophobicity, or steric properties). This alters the binding affinity and specificity of Cas9 for target sequences, reducing off-target effects while maintaining on-target activity.
2Manufacturing precision
If gRNA with low non-target gene candidates is selected, then target specificity is improved, but the range of applicable target genes is limited
Solution Approach 1:
The patent creates a universal SpCas9 variant with improved specificity that can be applied across multiple different target genes and genomic loci. The engineered Cas9 protein maintains broad applicability while consistently reducing off-target effects, making it a versatile tool for various genome editing applications.
Data Source
AI summary
The present invention relates to an artificially engineered CRISPR/Cas9 system. More particularly, the present invention relates to an artificially engineered CRISPR enzyme having enhanced target specificity and a use of an artificially engineered CRISPR/Cas9 system including the same enzyme in genome and/or epigenome manipulation or modification, genome targeting, genome editing, and in vitro diagnosis, etc.