Engineered Nuclease Specificity via Charge Mutation
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Solution Overview
Problem
Current artificial nuclease systems face challenges in achieving high specificity for targeted genomic modifications, often resulting in off-target cleavage due to excess nuclease activity and differing binding affinities of monomeric components, which can lead to decreased overall specificity and increased off-target activity.
Innovation Solution
Engineered nuclease cleavage systems with mutations in DNA binding domain regions and FokI nuclease cleavage domains are developed to enhance specificity, including mutations that alter charge and interactions with the DNA backbone, and independent titration of cleavage half-domain partners to optimize their ratios for reduced off-target activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional artificial nuclease systems are used, then genomic cleavage activity is achieved, but off-target cleavage increases due to excess nuclease activity and differing binding affinities
Solution Approach 1:
The patent applies parameter changes by mutating amino acid residues in the FokI nuclease domain to alter its biochemical properties. Specifically, positively charged residues (lysine and arginine) that interact with the DNA backbone are mutated to neutral or negatively charged residues, thereby changing the electrostatic interactions and reducing non-specific DNA binding. This parameter change in the nuclease's charge distribution directly reduces off-target cleavage while preserving on-target activity.
Solution Approach 2:
The patent applies local quality by making site-specific mutations at particular positions within the FokI nuclease domain rather than uniform modifications throughout the protein. The mutations are localized to specific residues (e.g., positions 416, 422, 447, 448, 525) that are known to interact with DNA phosphates, thereby locally modifying the interaction properties only where needed to reduce non-specific binding without affecting overall nuclease function.
2Productivity
If nuclease concentration is increased to enhance on-target cleavage, then cleavage efficiency improves, but off-target activity increases due to excess nuclease
Solution Approach 1:
The patent changes the biochemical parameters of the nuclease itself rather than adjusting operational parameters like concentration. By mutating residues that mediate non-specific DNA interactions, the nuclease's affinity for off-target sequences is reduced, allowing higher concentrations to be used for on-target cleavage without proportionally increasing off-target activity.
3Power
If conventional FokI nuclease domain is used, then cleavage activity is maintained, but non-specific interactions with DNA backbone phosphates occur
Solution Approach 1:
The patent changes the electrostatic parameters of the FokI nuclease domain by mutating positively charged residues to neutral or negatively charged residues. This alters the charge distribution and electrostatic interactions with the negatively charged DNA backbone, reducing non-specific binding while preserving the catalytic function of the nuclease.
Solution Approach 2:
The patent converts the harmful non-specific electrostatic interactions into a beneficial property by reducing them through mutation. The positively charged residues that cause non-specific DNA backbone binding are mutated, thereby eliminating the harmful interaction while the nuclease retains its specific cleavage activity at target sites.
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 engineered nuclease systems demonstrate significant increases in on-target specificity and reductions in off-target cleavage activity, with methods providing up to 50-200% increase in on-target cleavage and 1-1000-fold decrease in off-target activity, depending on the specific mutations and titration ratios used.
Implementation Method 1
Intermolecular ion pairs (salt bridges) are essential for many DNA-protein interactions. Often, charged amino acid side chains (i.e. —NH3+, =NH2+) interact with the negatively charged phosphate groups of the DNA backbone to form a salt bridge.
Implementation Method 2
The arginine at this position can interact with a phosphate on the DNA backbone via formation of a charged hydrogen bond with its side-chain guanidinium group.
Data Source
AI summary
Described herein are engineered nucleases comprising mutations in the cleavage domain (e.g., FokI or homologue thereof) and/or DNA binding domain (zinc finger protein, TALE, single guide RNA) such that on-target specificity is increased.