Cas9 Variants with Disrupted Linkers for Precise Genome Editing
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Solution Overview
Problem
Current Cas9 proteins exhibit high RuvC and HNH cleavage activities, leading to non-specific DNA cleavage, which can be detrimental in genome editing applications where precision is crucial.
Innovation Solution
Development of variant Cas9 proteins with disrupted RuvC/HNH linker regions or deletions within the HNH domain, reducing cleavage activity to nickase levels, and incorporating heterologous amino acid sequences for modified activities such as transcription modulation or nucleic acid modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cas9 protein is used with high RuvC and HNH cleavage activities, then DNA cleavage efficiency is improved, but non-specific DNA cleavage occurs reducing precision
Solution Approach 1:
The Cas9 protein is divided into separate functional domains: the RuvC domain and HNH domain are separated by a disrupted linker region. This segmentation allows independent control of cleavage activities, enabling the protein to perform site-specific nicking without non-specific double-stranded cleavage, thus resolving the contradiction between efficiency and precision.
Solution Approach 2:
The linker region between RuvC and HNH domains is locally modified through deletion or substitution of specific amino acids (e.g., residues 915-937). This local quality change reduces RuvC cleavage activity specifically while preserving HNH-mediated site-specific cleavage, achieving precise DNA modification without non-specific damage.
2Manufacturing precision
If RuvC/HNH linker region is disrupted to reduce RuvC cleavage activity, then DNA cleavage precision is improved, but cleavage efficiency decreases
Solution Approach 1:
Instead of completely abolishing RuvC activity, the disrupted linker region partially reduces RuvC cleavage activity to nickase levels. This partial action maintains sufficient cleavage efficiency for genome editing while eliminating non-specific double-stranded cleavage, achieving the desired balance between precision and productivity.
3Adaptability or versatility
If heterologous amino acid sequences are incorporated into Cas9, then functional versatility is improved, but protein structure complexity increases
Solution Approach 1:
The Cas9 protein is engineered to perform multiple functions: site-specific DNA cleavage via HNH domain, transcriptional regulation through disrupted RuvC activity, and potential fusion with heterologous domains. This multi-functionality is achieved through modular domain architecture where the disrupted linker region serves as a platform for additional functional modules, resolving the contradiction between versatility and complexity.
Data Source
Figure 1A~1B
Figure 2
Figure 3A~3B
AI summary
The present disclosure provides variant Cas9 proteins, nucleic acids encoding the variant Cas9 proteins, and host cells comprising the nucleic acids. The present disclosure provides systems that include a subject variant Cas9 protein (and/or a nucleic acid encoding the variant Cas9 protein) and a Cas9 guide RNA. In some cases, a subject system includes a PAMmer and/or a donor polynucleotide. The variant Cas9 proteins and the nucleic acids encoding the variant Cas9 proteins are useful in a wide variety of methods, which are also provided. In some embodiments, a variant Cas9 protein includes a RuvC domain, an HNH domain, and a disrupted RuvC/HNH linker region that reduces the RuvC cleavage activity of the protein. In some embodiments, a variant Cas9 protein includes a deletion (of all or a part of the HNH domain) or an insertion (within the HNH domain) that reduces the HNH cleavage activity of the protein.