Cas9 Variants Enhance On-Target Activity and Specificity
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Solution Overview
Problem
Existing CRISPR/Cas9 systems suffer from low activity and high frequency of off-target cleavage, limiting their effectiveness in therapeutic and research applications.
Innovation Solution
Engineering of SpCas9 variants using computational mutagenesis of the HNH domain, combined with a rapid yeast screening system, to enhance on-target activity and mutagenesis rates, specifically replacing amino acid residues in defined regions of the Cas9 protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If Cas9 mutations are introduced to lower off-target cleavage, then off-target cleavage frequency is reduced, but on-target activity and mutagenesis rate decrease
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (Q646, N749, T840, T846, N971) in the Cas9 protein to alter its catalytic properties. These parameter changes in the protein sequence result in variants with modified on-target activity and off-target cleavage characteristics, resolving the contradiction between reducing off-target effects and maintaining mutagenesis rate
Solution Approach 2:
The patent applies local quality by introducing specific mutations at defined positions within the Cas9 protein structure, particularly in regions that contact guide RNA or DNA. By modifying local amino acid properties at these specific sites while leaving the rest of the protein unchanged, the patent achieves selective improvement in on-target activity without uniformly affecting the entire protein
2Productivity
If Cas9 activity is increased to improve gene editing efficiency, then mutagenesis rate increases, but off-target cleavage frequency also increases
Solution Approach 1:
The patent uses parameter changes by modifying specific amino acid residues (particularly at positions Q646, N749, T840, T846, N971) to alter the Cas9 protein's catalytic parameters. These changes enable the protein to achieve higher on-target activity while simultaneously reducing off-target cleavage, thus resolving the contradiction between editing efficiency and specificity
Solution Approach 2:
The patent applies dynamics by creating Cas9 variants with modified conformational dynamics or catalytic cycle kinetics. The mutations introduce dynamic changes in how the protein interacts with DNA and guide RNA during the cleavage process, enabling faster or more efficient on-target cleavage while maintaining stringency that prevents off-target effects
3Reliability
If wild-type Cas9 is used to maintain natural specificity, then off-target cleavage is controlled, but on-target activity is insufficient for therapeutic applications
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid parameters (charge, hydrophobicity, size) at specific positions in the Cas9 protein. These parameter modifications create variants that maintain or improve specificity while enhancing on-target activity, overcoming the limitations of wild-type Cas9 for therapeutic applications
Solution Approach 2:
The patent applies composite materials by creating hybrid Cas9 variants that combine elements of wild-type specificity with engineered activity enhancements. The variants integrate specific mutant residues into the wild-type backbone, creating a composite protein structure that exhibits both high specificity and enhanced on-target activity
Data Source
AI summary
Provided herein are Cas9 proteins comprising SEQ ID NO:1 or a sequence at least 80% identical thereto, wherein: the amino acid residues at positions 765 to 780 are replaced by the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; the amino acid residues at positions 838 to 853 are replaced by the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10; and/or the amino acid residues at positions 911 to 925 are replaced by the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13. Also provided are Cas9 proteins comprising an HNH domain comprising the amino acid sequence of SEQ ID NO:14 or a sequence at least 80% identical thereto, wherein: the amino acid residues at positions 1 to 16 are replaced by the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; the amino acid residues at positions 74 to 89 are replaced by the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10; and/or the amino acid residues at positions 147 to 161 are replaced by the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.