Engineered SpCas9 Variants for Reduced Off-Target Editing
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Solution Overview
Problem
Current genome editing tools, such as CRISPR/Cas systems, suffer from off-target editing activity, which can lead to unintended genetic modifications and reduce the safety and efficacy of genome editing therapies.
Innovation Solution
Engineered Streptococcus pyogenes Cas9 (SpCas9) variants with specific amino acid substitutions, such as at positions 924, 929, and 930, are developed to enhance target specificity, reducing off-target editing while maintaining on-target activity when used in CRISPR/Cas endonuclease systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If wild-type SpCas9 is used in CRISPR/Cas system, then on-target editing activity is achieved, but off-target editing activity occurs reducing precision
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions 924, 929, and 930 in the SpCas9 protein sequence. These substitutions modify the biochemical parameters of the Cas9 protein to enhance its discrimination between on-target and off-target DNA sequences, thereby improving target specificity while maintaining editing activity.
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (924, 929, 930) within the SpCas9 protein rather than modifying the entire protein. These localized changes at the PAM interaction region and other critical domains improve target specificity without compromising the overall function and on-target editing capability of the Cas9 protein.
2Measurement precision
If amino acid substitutions are introduced to reduce off-target activity, then target specificity is improved, but on-target editing activity may be reduced
Solution Approach 1:
The patent optimizes parameter changes by selecting specific amino acid substitutions that improve target specificity while preserving catalytic function. The substitutions at positions 924, 929, and 930 are carefully chosen to modify DNA binding specificity without affecting the nuclease active site, thereby maintaining on-target editing productivity.
Solution Approach 2:
The patent applies local quality by restricting modifications to specific regions of the SpCas9 protein that are involved in DNA recognition and binding, while leaving the catalytic domains intact. This ensures that on-target editing activity is preserved while improving target specificity through localized sequence optimization.
Data Source
AI summary
The present invention is directed to, inter glia, composition and methods for genome editing. Specifically, a non-naturally occurring SpCas9 variant having an amino acid substitution at position K929, H930, or at both position K929 and H930.