Engineered SpCas9 Mutations for High-Specificity Genome Editing
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Solution Overview
Problem
Existing CRISPR-Cas systems suffer from low target specificity, leading to unintended modification of non-target genes or nucleic acids due to partial complementary binding, necessitating improved methods to enhance target gene or nucleic acid modification efficiency.
Innovation Solution
A Streptococcus pyogenes Cas9 (SpCas9) variant with specific amino acid substitutions or deletions in key regions, such as A203D, N277H, G366S, F539S, I601N, M763I, D965Y, F1038Y, K890N, and D1127E, to enhance target specificity and reduce off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the CRISPR-Cas system is used to modify target genes or nucleic acids, then genome editing efficiency is improved, but off-target effects occur leading to modification of non-target genes or nucleic acids
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues in the SpCas9 protein (e.g., N277H, A203D, G366S substitutions) to alter its binding affinity and cleavage activity. These parameter changes in the enzyme's structure enable it to distinguish between perfectly complementary target sites and partially complementary off-target sites, thereby reducing off-target effects while maintaining on-target editing efficiency.
Solution Approach 2:
The patent applies local quality by introducing specific mutations at particular amino acid positions (such as positions 277, 203, 366, 539, 601, 763, 965, 1038, 890, and 1127) that are located in specific functional regions of the Cas9 protein. These localized changes affect the enzyme's interaction with DNA at specific sites, enhancing its ability to recognize and cleave only the intended target sequence while leaving similar but non-identical sequences unaffected.
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 achieves universality by engineering a modified SpCas9 variant with improved specificity that can be applied across a broad range of different gRNA targets. The enzyme's enhanced discrimination capability allows it to work effectively with various gRNA sequences, expanding the range of applicable target genes beyond what is possible with wild-type Cas9, thus providing both high specificity and broad versatility.
Solution Approach 2:
By changing the biochemical parameters of the Cas9 enzyme through amino acid substitutions, the patent enables the enzyme to maintain high target specificity across diverse gRNA sequences. This parameter change in the enzyme's properties allows it to accommodate a wider variety of target genes while preserving the ability to distinguish perfect matches from partial matches, thereby increasing both precision and adaptability.
Data Source
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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.