Orthogonal Base Editing via Aptamer-gRNA Tethering
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Solution Overview
Problem
Current genome editing methods, particularly those using CRISPR-Cas9, suffer from high rates of unwanted gene alterations and low efficiencies when multiplexing point mutation introduction, especially when multiplexing point mutations, and are impractical for multiplexing due to high cytotoxicity and undesired editing outcomes, such as translocations, large-scale deletions, and chromosomal aberrations.
Innovation Solution
Aptamer-based multiplexed orthogonal base editor (MOBE) systems that utilize RNA aptamers to engineer simultaneous introduction of C⋅G to T⋅A and A⋅T to G⋅C SNVs at distinct protospacers with minimal crosstalk, employing a single Cas enzyme and RNA-based delivery methods, combining an aptamer-gRNA construct with a coat protein fusion, and a coat protein to tether the enzymes directly to their respective gRNAs, and a coat protein-deaminase fusion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If DSB-reliant genome editing methods (Cas9) are used to introduce point mutations, then precise editing at target sites is achieved, but high rates of unwanted gene alterations (indels, translocations, large-scale deletions) and cytotoxicity occur, making multiplexing impractical
Solution Approach 1:
The invention divides the single Cas9 enzyme function into two separate enzymes: Cas9 nickase (which creates single-strand breaks) and a separate deaminase enzyme (which performs the base conversion). This segmentation allows each enzyme to perform its specific function without causing the harmful effects associated with double-strand breaks, thereby enabling precise point mutation introduction without indels or chromosomal aberrations.
Solution Approach 2:
The invention extracts the harmful double-strand break function from the editing process. By using Cas9 nickase to create only single-strand breaks and relying on the cell's base excision repair pathway instead of HDR or NHEJ, the method eliminates the generation of indels, translocations, and large-scale deletions that normally accompany DSB repair.
2Productivity
If multiple DSBs are introduced simultaneously for multiplexing, then multiple point mutations can be introduced, but success rates decrease exponentially and incidences of translocations, large-scale deletions, chromosomal aberrations, and p53-mediated apoptosis increase
Solution Approach 1:
The invention segments the editing function across multiple gRNAs directed to different target sites, with each gRNA-Cas9 nickase-deaminase complex acting independently. This allows simultaneous introduction of multiple point mutations without the cumulative cytotoxicity and chromosomal damage associated with multiple DSBs, as each site undergoes gentle single-strand break and base conversion rather than dangerous double-strand break repair.
Solution Approach 2:
The invention introduces the deaminase enzyme as an intermediary that performs the actual base conversion chemistry. This intermediary approach allows the system to achieve point mutation introduction without relying on error-prone DSB repair pathways, thereby maintaining high cell viability and editing accuracy even when multiplexing at multiple genomic loci.
3Object-generated harmful factors
If base editors are used for point mutation introduction, then DSB-free editing with reduced cytotoxicity is achieved, but multiplexing CBEs and ABEs together faces challenges due to gRNA crosstalk
Solution Approach 1:
The invention segments the gRNA structure by inserting aptamer sequences at specific locations within the gRNA backbone. This segmentation creates distinct molecular identifiers that allow different gRNAs to be selectively recognized by their corresponding deaminase enzymes, preventing crosstalk while maintaining the simplified single-strand break mechanism of base editors.
Solution Approach 2:
The invention uses aptamer-deaminase interactions as intermediaries to ensure selective enzyme-gRNA pairing. The aptamers serve as molecular mediators that specifically recruit the intended deaminase enzyme to its corresponding gRNA, preventing off-target recruitment and crosstalk between different base editing systems while maintaining the low cytotoxicity advantage of DSB-free editing.
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 MOBE systems achieve up to 25.3% co-occurring orthogonal edits with crosstalk rates of only 1.1%, significantly improving editing precision and reducing unwanted byproducts.
Implementation Method 1
Aptamer-based multiplexed orthogonal base editor (MOBE) systems that utilize RNA aptamers to engineer simultaneous introduction of C⋅G to T⋅A and A⋅T to G⋅C SNVs at distinct protospacers with minimal crosstalk
Implementation Method 2
The Cas9 enzyme complexes with a guide RNA (gRNA) molecule, which directs the fusion protein to the target site (called the protospacer) via base-pairing rules
Implementation Method 3
The Cas9:gRNA:DNA ternary complex is an R-loop, in which one DNA strand is base-paired with the gRNA, and the other is single-stranded and lacks a complement
Implementation Method 4
where the ssDNA modifying enzyme directly chemically modifies target nucleotides within this window (FIG. 1A—prior art). Two major classes of base editors have been developed that use cytosine and adenine deamination chemistries to catalyze the conversion of C⋅G base pairs to T⋅A (CBEs), and A⋅T base pairs to G⋅C (ABEs), respectively
Data Source
AI summary
The base editor systems (“MOBE”) that are derived from the CRISPR/Cas9 protein that enable to simultaneously introduce C⋅G to T⋅A and A⋅T to G⋅C point mutations at distinct genomic loci in living cells, with high efficiency and precision. In the MOBE disclosed herein, a piece of RNA (“the gRNA”) of the CRISPR/Cas9 protein is fused to the deaminase enzymes via a coat protein-aptamer interaction. A reporter plasmid comprising the MOBE system that allows for enrichment of cells with co-occurring orthogonal edits and increased editing efficiency.