Non-covalent Fusion Proteins for Precise Genomic Editing
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Solution Overview
Problem
Current gene editing technologies face challenges in achieving precise and efficient localization of proteins to specific genomic loci for targeted gene modification, particularly in gene editing applications where DNA binding domains fused with nuclease domains do not provide sufficient efficacy.
Innovation Solution
The development of fusion proteins comprising a DNA localization component, such as guide RNAs or DNA-binding domains from TALENs, combined with effector molecules like Cas9 or Type IIS endonucleases, which can be linked via non-covalent linkages to operate at specific genomic loci, enabling targeted DNA cutting and modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA binding domains are fused to nuclease domains via covalent linkage for gene editing, then the protein can be localized to specific genomic loci, but the nuclease activity and editing efficiency are insufficient
Solution Approach 1:
The fusion protein is divided into two separate components: a DNA binding component (such as zinc finger protein, TALE, or guide RNA-Cas9 complex) and a nuclease component (such as FokI or Cas9). These components bind to the target DNA sequence and execute cutting function respectively, rather than being covalently fused. This segmentation allows each component to function optimally while achieving reliable gene editing through their coordinated action at the target locus.
2Productivity
If covalent fusion is used to link DNA binding domain and nuclease domain, then the protein structure is simplified, but the cutting efficiency at target locus is reduced
Solution Approach 1:
An intermediary mechanism is introduced to link the DNA binding component and nuclease component. This intermediary can be a peptide linker in covalent fusion designs, or a protein-protein interaction interface in non-covalent assemblies. The intermediary allows the two components to maintain proper spatial relationship and communication while preserving the high cutting efficiency of the nuclease at the target locus, avoiding the efficiency loss associated with direct covalent fusion.
3Reliability
If non-covalent linkages are used to connect DNA localization component and effector molecule, then nuclease activity is enhanced, but the stability of the complex may be reduced
Solution Approach 1:
The non-covalent complex exhibits local quality differentiation: the DNA binding component provides stable and specific binding to the target DNA sequence, while the nuclease component maintains high catalytic activity and flexibility. This local quality distribution allows the complex to achieve both stable targeting and enhanced nuclease activity, as each component optimizes its function in its local environment rather than being constrained by a rigid covalent structure.
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
This approach provides superior and unexpectedly efficient nuclease activity at target loci, allowing for precise gene editing by interrupting gene expression or modifying genomic sequences through insertion, deletion, or substitution of base pairs, demonstrating enhanced cutting efficiency compared to existing methods.
Implementation Method 1
a first gRNA specifically binds to a first strand of a double-stranded DNA target sequence and a second gRNA specifically binds to a second strand of a double-stranded DNA target sequence
Implementation Method 2
one or more strand of the double-stranded DNA may be cut... Compositions and methods of the disclosure provide superior and unexpectedly efficient nuclease activity at a target locus or loci in a genome
Data Source
AI summary
Disclosed are compositions and methods for directing proteins to specific loci in the genome and uses thereof. In one aspect, the disclosed methods allow for directing proteins to specific loci in the genome of an organism, including the steps of providing a fusion protein comprising a DNA localization component and an effector molecule. Preferred embodiments of the disclosure include, but are not limited to, the following fusion proteins: dSaCas9-Clo051, dCas9-Clo051, Xanthomonas-TALE-Clo051, and Ralstonia-TALE-Clo051.