Truncated Cas9 Variants for Viral Packaging Constraints
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Solution Overview
Problem
The large size of Cas9 proteins limits their potential for biotechnology and therapeutic applications due to difficulties in delivery and manipulation, particularly in biotechnological and therapeutic contexts where gene size constraints, such as viral packaging limits, hinder efficient use.
Innovation Solution
Engineering RNA-guided DNA binding proteins by identifying and removing non-conserved regions within the Cas9 protein, creating smaller variants that retain near wild-type activity, allowing for targeted DNA binding and editing while reducing gene size for more efficient delivery and manipulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the full-length Cas9 protein is used, then DNA binding and nuclease activity are maintained, but the protein size becomes too large for efficient delivery and manipulation
Solution Approach 1:
The patent extracts and removes non-essential domains and regions from the full-length Cas9 protein to create truncated variants. Specifically, the patent deletes the RuvC domain (nuclease domain) to create Cas9 nickases, and further removes additional regions to create even smaller variants. This extraction of unnecessary components reduces protein size while preserving the essential DNA binding function mediated by the guide RNA complex.
Solution Approach 2:
The patent segments the Cas9 protein into functional domains, identifying and preserving only the essential regions required for DNA binding while removing non-essential segments. The protein is divided into functional modules: the guide RNA binding region, the PAM recognition region, and the DNA binding region are preserved, while the RuvC nuclease domain and other non-essential regions are segmented out and removed.
2Ease of operation
If the Cas9 protein size is reduced, then delivery and manipulation efficiency improve, but protein functionality may be compromised
Solution Approach 1:
The patent applies local quality by creating different variants of Cas9 with specific functional properties tailored to different applications. Some variants retain full nuclease activity (Holliday junction endonuclease activity) while others are engineered as nickases (single-strand cutting) or dominant-negative variants. Each variant has locally optimized properties: smaller size for delivery, preserved DNA binding for targeting, and controlled nuclease activity for specific applications.
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 resulting smaller Cas9 variants can efficiently bind to DNA, create double-stranded breaks, or function as nuclease-null proteins, enhancing their delivery and functionality in both prokaryotic and eukaryotic cells, including human cells, thereby overcoming size limitations and improving biotechnological and therapeutic applications.
Implementation Method 1
Cas9 is a DNA nuclease that can be programmed to target nearly any region of a genome by expressing a guide RNA (gRNA) that contains a motif that recruits Cas9 and 20 basepairs of complementarity to a region of the genome where targeting is desired
Implementation Method 2
functioning as an RNA guided DNA binding nuclease that can bind to target DNA and create a double stranded break in target DNA
Implementation Method 3
functioning as an RNA guided DNA binding nickase that can bind to target DNA and create a single stranded break or nick in target DNA
Data Source
AI summary
Methods of making mutant Cas9 proteins are described.