Cas9 Polypeptide Mutations for Reduced Off-Target Editing
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Solution Overview
Problem
Current CRISPR-Cas9 systems suffer from significant off-target editing activities while maintaining robust on-target editing efficiency, necessitating the development of polypeptides with reduced off-target effects.
Innovation Solution
Development of CRISPR polypeptides, such as Cas9 and Cas12, with specific amino acid mutations at positions Y256, R314, and Q414 or R654, which reduce off-target activity while preserving on-target efficiency, and their use in vectors like AAV for in vivo genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type SaCas9 is used, then robust on-target editing activity is achieved, but significant off-target editing activity occurs
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (Y256A, R314A, Q414A, R654A) at defined positions in the SaCas9 polypeptide sequence. These mutations alter the binding affinity and specificity parameters of the Cas9 protein, reducing off-target editing activity while maintaining on-target editing activity through controlled changes in the protein's structural and functional properties
Solution Approach 2:
The patent applies local quality by making targeted mutations at specific positions (Y256, R314, Q414, R654) within the Cas9 polypeptide sequence. Each mutation is localized to a specific region, allowing precise modification of binding characteristics at key interaction sites while preserving the overall functionality and on-target activity of the Cas9 enzyme
2Object-generated harmful factors
If Cas9 polypeptide mutations are introduced to reduce off-target activity, then off-target editing is reduced, but on-target activity may be compromised
Solution Approach 1:
The patent applies partial action by introducing a specific set of mutations (Y256A, R314A, Q414A, R654A) that provides sufficient off-target reduction without over-modifying the protein. This balanced approach ensures that the mutations are enough to achieve the desired specificity improvement while maintaining adequate on-target activity through controlled, partial modification rather than extensive changes
Data Source
AI summary
The present invention relates to CRISPR polypeptides, including Cas9 and Cas12 5 polypeptides, with reduced off-target activity. The invention also provides compositions comprising such polypeptides, vectors encoding such polypeptides and methods of using such polypeptides.