Methods for exon skipping and gene knockout using base editors
Inventors
Perez-Pinera, Pablo • GAPINSKE, Michael P. • WINTER, Jackson Scott
Assignees
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Publication Number
US-12612612-B2
Publication Date
2026-04-28
Expiration Date
Abstract
The disclosure provides a versatile method termed CRISPR-SKIP that utilizes cytidine and/or adenine deaminase base editors to program exon skipping by mutating target DNA bases within splice acceptor sites and/or splice enhancer sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.
Core Innovation
The invention relates to inducing selective exon skipping by contacting one or more DNA target sequences with a single guide RNA (sgRNA) and a fusion protein that includes at least two tRNA-specific adenosine deaminase (TadA) domains, a linker, and an RNA-guided DNA endonuclease having nickase activity capable of causing single stranded breaks in the DNA target sequences, such that an exon is selectively skipped. The document describes CRISPR-SKIP and adenine base editor-mediated splice disruption in splice acceptor site context.
The system combines nickase activity with TadA deaminase domains to program exon skipping without causing double stranded breaks. The targeting is directed by the sgRNA to specific DNA target sequences, which are described in the dependent claims as splice acceptor regions and/or splice enhancer regions, with PAM-dependent protospacer positioning relative to the intended splice region.
The document also describes recombinant architectures for the fusion protein, including configurations in which the components are provided in first and second constructs and linked using inteins to form a fusion protein. Split versions of the RNA-guided DNA endonuclease nickase activity are described, including N-terminal and C-terminal intein arrangements, optional inclusion of UGI, and delivery concepts using adeno-associated virus (AAV) vectors, including packaging of split constructs.
Claims Coverage
The document provides one independent claim directed to inducing selective exon skipping, with dependent claims that refine targeting features, fusion construction elements, and delivery constraints.
Selective exon skipping with sgRNA and TadA-linker fusion to nickase endonuclease
A method for inducing selective exon skipping comprising contacting one or more DNA target sequences with an sgRNA complementary to the one or more DNA target sequences and a fusion protein comprising at least two tRNA-specific adenosine deaminases (TadA) domains, a linker, and an RNA-guided DNA endonuclease having nickase activity capable of causing single stranded breaks in the one or more DNA target sequences, such that an exon is selectively skipped.
Targeting splice acceptor or splice enhancer regions of named genes/exons
The method includes selecting the one or more DNA target sequences from splice acceptor or splice enhancer regions of alpha-synuclein, dystrophin, and Huntington genes/exons.
TadA domain provenance from specified bacterial sources
The method specifies that the at least two TadA domains are obtained from Escherichia coli, Bacillus subtilis, or Staphylococcus aureus.
Split fusion protein encoding with N-terminal and C-terminal inteins
The method includes forming a fusion protein by encoding it with a first and a second construct containing tRNA-specific adenosine deaminases (TadA), a linker, and split RNA-guided DNA endonuclease nickase activity fused to N-terminal and C-terminal inteins.
Defined linker peptide sequences for connecting fusion components
The method specifies that the linker comprises one of several defined peptide sequences, including (AP)5 (SEQ ID NO:2), GGGGS (SEQ ID NO:3) and (GGGGS)n variants, a longer SEQ ID NO:10 sequence, or (EAAA)5 (SEQ ID NO:11).
AAV packaging of split constructs
The method includes packaging the first and second constructs into first and second adeno-associated virus (AAV) vectors.
Overall, the claims coverage centers on a selective exon skipping method using an sgRNA-directed RNA-guided DNA endonuclease nickase combined with a TadA-containing fusion protein, with additional limitations on splice acceptor/enhancer targeting, TadA source, split fusion architecture via inteins, allowable linker sequences, and AAV packaging of split constructs.
Stated Advantages
Expands exon-skipping targets beyond BE3 PAM constraints by disrupting splice acceptor sites using adenine base editors.
Achieves selective exon skipping associated with A>G editing at splice-directed target contexts.
Provides genome-wide evidence that ABE targetability of inner exons exceeds BE3 across efficiency and specificity thresholds.
Reports low off-target mutation rates based on high-throughput sequencing assessment.
Simplicity and precision of the programmable exon skipping approach.
Claims report high exon-skipping rates and off-target profiles.
Documented Applications
Therapeutic exon skipping in disease-relevant genes, including SNCA exon 3 skipping using full-length and split ABE with efficiency analysis.
Caspase-6 cleavage-site modification via splicing in Huntington, described using splicing-driven modification at an exon 12 or 13 region using SBE technology.
Dystrophin (DMD) exon 45 skipping in myoblasts using ABE GGGGS-UGI, split ABE GGGGS-UGI, SpBE3, and split SpBE3 with reported skipping rates.
Splice-directed exon skipping targeting AHCY exon 9 using AAV split ABE components with A>G editing and exon skipping measurements in HEK293T cells.
Programmable skipping of RELA exon 7 and PIK3CA exon 5.
Disease-relevant exon skipping examples including SNCA (Parkinson’s disease), HTT (Huntington disease), and DMD exon 45 skipping (Duchenne muscular dystrophy).
Genome-wide computational targetability via a CRISPR-SKIP web tool and predicted exon coverage.
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