Cas9 Enzyme Mutations Enhancing Spacer Acquisition and NAG Cleavage
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Solution Overview
Problem
Current Cas9 systems have limitations in spacer acquisition and target cleavage efficiency, particularly with NAG PAMs, necessitating improvements for enhanced CRISPR-Cas immunity and bacterial protection against bacteriophages.
Innovation Solution
Development of novel Cas9 enzymes with mutations at specific amino acid positions, such as I473 and K500, which enhance spacer acquisition and cleavage efficiency, specifically targeting NAG PAMs, leading to improved CRISPR-Cas immune responses and increased resistance to bacteriophages.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type Cas9 is used, then the system maintains natural specificity for NGG PAMs, but spacer acquisition rate and cleavage efficiency are limited
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the Cas9 protein sequence (e.g., positions 473, 500, 756, 1032) to alter its functional properties. These mutations change the biochemical parameters of Cas9, enabling enhanced spacer acquisition rates and improved cleavage efficiency while maintaining or expanding PAM recognition beyond the wild-type NGG specificity.
Solution Approach 2:
The invention applies local quality by making targeted mutations at specific positions within the Cas9 protein structure rather than global modifications. The mutations at particular amino acid positions (such as 473, 500, 756, 1032) locally modify the protein's interaction surfaces, allowing enhanced function at specific interfaces while preserving overall protein structure and function.
2Reliability
If Cas9 mutations are introduced to enhance spacer acquisition, then immunity response is improved, but protein structure complexity increases
Solution Approach 1:
The patent introduces specific amino acid substitutions at defined positions in the Cas9 sequence to change its functional parameters. These controlled parameter changes enhance spacer acquisition and cleavage efficiency without requiring complex structural redesign, maintaining relative simplicity while improving immune response effectiveness.
3Adaptability or versatility
If Cas9 is used for NGG PAM targets, then cleavage efficiency is maintained, but it cannot effectively target NAG PAMs
Solution Approach 1:
The patent achieves universality by engineering Cas9 variants that can recognize multiple PAM types (NGG, NAG, and potentially others). The mutations enable a single Cas9 protein to perform multiple target recognition functions, expanding the adaptability of the CRISPR system to diverse genomic targets while maintaining productive cleavage across different PAM contexts.
Solution Approach 2:
Through amino acid mutations at key positions, the patent changes the PAM recognition parameters of Cas9, allowing it to accommodate different PAM sequences (NGG, NAG, etc.). This parameter modification enables broader target range without sacrificing cleavage efficiency, as the mutated Cas9 maintains its catalytic function while gaining enhanced PAM flexibility.
Data Source
AI summary
Provided are Cas9 enzymes that have mutations that enhance their properties, relative to un-mutated Cas9. The altered Cas9 enzymes exhibit i) an increased rate of spacer acquisition, or ii) increased cleavage efficiency of targets with NAG PAMs, or a combination of i) and ii). The altered Cas9 enzymes comprise an amino acid substitution of 1473 and K500 in a Streptococcus pyogenes or similar Cas9 enzyme. Also provided are polynucleotides, including expression vectors that encode the Cas9 enzymes, cells that contain the polynucleotides, and methods of making and using such cells. The disclosure includes tagging, or labelling bacteria, and for enhancing phage acquired immunity in bacteria, such as those used in industrial processes, including the food and beverage industry, such as the dairy industry. The food products are also included.