Fusion Effector Proteins for Precise Genome Editing
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Solution Overview
Problem
Current genome editing methods, such as CRISPR-associated nucleases, are inefficient for precise DNA or RNA modifications and often result in undesired byproducts when attempting to correct point mutations, which are common in genetic disorders.
Innovation Solution
Development of fusion effector proteins comprising base editing enzymes, prime editing enzymes, transcriptional activators, inhibitors, and transposases, which are fused with guide nucleic acids and linked via linkers, allowing for precise nucleobase modifications and gene expression modulation without generating double-stranded DNA breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-associated nucleases are used for genome editing, then DNA sequences can be precisely targeted and disrupted, but the method generates double-stranded DNA breaks that lead to undesired byproducts and reduce editing precision
Solution Approach 1:
The invention divides the traditional nuclease function into separate components: a catalytically inactive Cas protein (dCas9) that provides targeting capability without cleavage activity, and separate base editing enzymes (deaminases, polymerases) that perform the actual nucleotide modification. This segmentation eliminates DNA breakage while maintaining precise targeting through the guide RNA-dCas9 complex.
Solution Approach 2:
The invention extracts and removes the nuclease cleavage function from the Cas protein by introducing catalytic inactivating mutations (e.g., D10A, H840A, N864D/N867A). This extraction eliminates the harmful double-stranded DNA breaks while preserving the programmable targeting capability of the guide RNA-Cas complex for precise base editing.
2Manufacturing precision
If base editing is used to correct point mutations, then precise nucleotide changes can be achieved, but the efficiency is limited compared to nuclease-based methods
Solution Approach 1:
The invention merges multiple functional domains into a single fusion protein: the catalytically inactive Cas protein for targeting, linkers for structural connection, and base editing enzymes (deaminases like ABE8e, TadA for transversion; or polymerases for insertion/deletion) for catalysis. This merging creates a unified base editor that combines precise targeting with efficient catalytic activity, significantly improving editing efficiency while maintaining accuracy.
Solution Approach 2:
The base editor functions as a composite molecular system combining proteins with different functions (dCas9 for binding, deaminase for chemical modification, polymerase for repair synthesis) into a single functional unit. This composite structure allows the editor to perform multiple operations (targeting, deamination, DNA repair) in a coordinated manner, enhancing overall editing efficiency.
3Adaptability or versatility
If fusion proteins with multiple functional domains are created, then versatile genome editing capabilities are achieved, but the device complexity increases
Solution Approach 1:
The invention introduces flexible linker sequences (e.g., (GGGGS)n, (Gly-Ser-Gly-Ala)n) between functional domains that provide dynamic flexibility and conformational adaptability. These linkers allow the fusion protein to adopt optimal spatial arrangements for each functional domain while maintaining overall structural integrity, effectively managing the complexity of multi-domain architectures.
Solution Approach 2:
The linker sequences serve as intermediary elements that connect and mediate between the dCas9 targeting domain and the base editing catalytic domain. These linkers facilitate proper spatial positioning and functional coordination between domains, simplifying the overall protein architecture by providing standardized connection modules that reduce design complexity.
Data Source
AI summary
The present disclosure provides compositions of CRISPR associated (Cas) effector proteins fused to partner proteins. Compositions typically comprise a guide nucleic acid. Also disclosed are the methods and systems for detecting and modifying target nucleic acids using the same. The cells, progenies thereof, and populations thereof produced by the compositions, methods, or systems provided herein are also described.