Cas9 Atomic Structures for Genome Engineering
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Solution Overview
Problem
There is a need to understand the structural basis for guide RNA recognition and DNA targeting by Cas9 enzymes, which are crucial for genome engineering but lack comprehensive structural insights.
Innovation Solution
The disclosure provides atomic structures of Cas9 with and without bound polynucleotides, along with computer-readable media containing atomic coordinates for Cas9 polypeptides in unbound and bound configurations, enabling the engineering of Cas9 polypeptides by identifying sites for amino acid residue substitution, insertion, or deletion to alter function, and the creation of chimeric Cas9 polypeptides for site-specific gene regulation and genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If atomic structures of Cas9 are determined to understand structural basis for guide RNA recognition and DNA targeting, then understanding of Cas9 mechanism is improved, but complexity of structural analysis and data interpretation increases
Solution Approach 1:
The patent creates physical crystal copies of Cas9 protein structures that can be analyzed and replicated. By crystallizing Cas9 with bound guide RNA and DNA, the invention produces tangible structural models that capture the molecular interactions, allowing researchers to study the structural basis without directly analyzing complex solution-state molecular dynamics.
Solution Approach 2:
The invention changes the physical state of Cas9 from soluble protein to crystalline form, and modifies chemical parameters by introducing specific mutations (e.g., D10A, H840A) to create catalytically inactive variants that can be crystallized. These parameter changes enable structural determination while preserving the binding interface of interest.
2Adaptability or versatility
If Cas9 polypeptides are engineered by amino acid residue substitution to alter function, then Cas9 activity is improved or modified, but structural stability may be compromised
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions at specific residues involved in catalytic activity (e.g., D10A, H840A in the HNH and RuvC domains) while leaving the rest of the protein structure intact. This localized modification alters Cas9 function (creating nickase or dead variants) without compromising overall structural stability, as the changes are confined to specific functional domains rather than the entire protein.
3Adaptability or versatility
If chimeric Cas9 polypeptides are created through domain replacement, then versatility of Cas9 is improved, but complexity of protein construction increases
Solution Approach 1:
The patent segments the Cas9 protein into functional domains (HNH domain, RuvC domain, PAM-interacting domain, guide RNA-binding domain) that can be independently manipulated. By dividing Cas9 into these modular segments, the invention enables domain replacement to create chimeric variants with altered specificities or functions, while the modular nature simplifies the construction process compared to attempting to redesign the entire protein.
Solution Approach 2:
The invention merges domains from different Cas9 orthologs or from other proteins into a single chimeric Cas9 polypeptide. For example, the patent combines PAM-interacting domains from different bacterial species to create Cas9 variants with expanded PAM recognition specificities, thereby increasing versatility through functional combination of complementary domains.
Data Source
Figure 1A~1B
Figure 2A
Figure 2B
AI summary
The present disclosure provides atomic structures of Cas9 with and without polynucleotides bound thereto. Also provided is a computer-readable medium comprising atomic coordinates for Cas9 polypeptides in both an unbound configuration and a configuration wherein the Cas9 polypeptide is bound to one or more polynucleotides. The present disclosure provides crystals comprising Cas9 polypeptides; and compositions comprising the crystals. The present disclosure provides methods for the engineering of Cas9 polypeptides wherein Cas9 activity has been altered, ablated, or preserved and amended with additional activities.