Mutant Cas Protein Engineering for Higher DNA Cleavage Activity
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Solution Overview
Problem
Current CRISPR/Cas systems have varying advantages and disadvantages in terms of guide RNA, PAM, and Cas protein size, necessitating the development of new Cas proteins and mutants to meet diverse application requirements.
Innovation Solution
A novel Cas protein and mutants thereof, including specific amino acid mutations at key sites, are developed to enhance cleavage activity, binding, and editing preference, with variants having up to 99.5% homology to the wild-type sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current CRISPR/Cas systems are used, then gene editing function is achieved, but cleavage activity and binding efficiency are limited
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues in the Cas protein sequence. Multiple mutants were generated with substitutions at positions such as S246, S286, C287, D288, E289, A290, I292, and other key sites. These parameter changes in the protein structure resulted in enhanced cleavage activity (up to 1000% increase) and improved binding efficiency, while maintaining the core gene editing function.
2Weight of moving object
If Cas protein size is reduced, then delivery efficiency improves, but cleavage activity may be compromised
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions at specific critical sites within the Cas protein rather than uniformly modifying the entire protein structure. Key residues such as S246, S286, C287, D288, E289, A290, I292, and others were selectively mutated to enhance cleavage activity. This localized modification approach allows optimization of specific functional regions while maintaining the overall protein size and structural integrity necessary for delivery efficiency.
3Reliability
If amino acid substitutions are made to enhance cleavage activity, then binding efficiency improves, but protein stability may be affected
Solution Approach 1:
The patent applies feedback by systematically testing multiple amino acid substitutions at key sites and selecting mutants that achieve the desired balance between enhanced binding efficiency and maintained protein stability. The process involved evaluating various mutants (e.g., S246A/S286F/C287I/D288W/E289A/A290V/I292L, S246R/S286F/C287I/D288W/E289A/A290V/I292L, and others) and iteratively optimizing the combination of mutations to achieve up to 1000% increase in cleavage activity while ensuring the protein remains stable and functional.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The Cas protein variants exhibit enhanced cleavage activity on target sequences, up to 1000% increase, and improved binding, addressing the limitations of existing systems.
Implementation Method 1
The Cas9 protein, with the assistance of a trans-encoded small RNA (tracrRNA), can process pre-crRNA into mature crRNA bound to the tracrRNA
Implementation Method 2
the spacer-specific endonuclease cleavage activity of the Cas protein variant on a target sequence of a target DNA complementary to the spacer is significantly increased
Data Source
Figure 1A
Figure 1B~2
Figure 3~4
AI summary
The present invention provides a Cas protein and its variants, and the corresponding gene-editing system and uses. Specifically, the Cas protein of the present invention has excellent gene-editing activity, can effectively edit or cleave a target gene, and can effectively treat disorders or diseases of a subject in need. The present invention also relates to an editing system and uses of a fusion protein containing the Cas protein or its variant.