Engineered Cleavage Half-Domains for Specific Genomic Targeting
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Solution Overview
Problem
Current zinc finger nucleases (ZFNs) face challenges in specificity due to off-target cleavage events, as they can form homodimers that lead to unintended DNA cleavage, reducing the precision of targeted genomic modifications.
Innovation Solution
Engineered cleavage half-domains that form obligate heterodimers with either wild-type or other engineered domains, minimizing homodimer formation and enhancing specificity by incorporating mutations at specific amino acid residues, such as E490K and I538K, are used in fusion proteins to facilitate targeted cleavage and homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If zinc finger nucleases are used for targeted cleavage of genomic DNA, then targeted mutagenesis and deletion can be achieved, but off-target cleavage events occur reducing specificity
Solution Approach 1:
The FokI cleavage domain is divided into two separate half-domains that must dimerize to become active. This segmentation ensures that cleavage only occurs when both zinc finger proteins bind to their respective target sites, preventing off-target cleavage by single zinc finger proteins
Solution Approach 2:
The patent introduces asymmetric mutations at the dimerization interface of the FokI half-domains. These mutations create heterodimeric specificity where only complementary half-domains (with matching mutations) can form functional dimers, preventing homodimer formation and ensuring precise targeted cleavage
2Reliability
If a pair of fusion proteins with zinc finger binding domains and cleavage half-domains is used, then specificity is increased, but homodimer formation can still occur leading to off-target cleavage
Solution Approach 1:
The patent introduces specific mutations at localized positions (483, 486, 487, 490, 499, 538) at the dimerization interface of the FokI half-domains. These local modifications create complementary surfaces that favor heterodimer formation while preventing homodimer formation, without affecting the overall structure or function of the cleavage domains
Solution Approach 2:
The patent alters the amino acid sequence parameters of the FokI half-domains by introducing point mutations at specific residues. These parameter changes modify the dimerization interface properties to enable obligate heterodimer formation, transforming the interaction specificity from promiscuous to highly selective
Data Source
AI summary
Disclosed herein are engineered cleavage half-domains; fusion polypeptides comprising these engineered cleavage half-domains; polynucleotides encoding the engineered cleavage half-domains and fusion proteins; and cells comprising these polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.