Abstract
Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently shown that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of CRISPR-Cas9 adenine deaminase base editors to disrupt the conserved adenine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9-nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon-skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress toward permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.
Highlights
Exon splicing is a natural process that occurs during mRNA maturation and results in exclusion of intronic sequences and the assembly of consecutive or nonconsecutive exons from pre-mRNA1
At AHCY exon 9 mutation rates of ∼20% in genomic DNA resulted in skipping rates of ∼50%, whereas at HSF1 exon 11, mutation rates of ∼50% in genomic DNA resulted in rates of exon skipping of only ∼20%
Since one of the major blocks during transcript elongation is the splicing junction[34,35,36], which leads to transient polymerase pausing at the splice sites[37], it is reasonable to expect that the rate of exon skipping can be higher than the conversion rate measured in genomic DNA
Summary
Exon splicing is a natural process that occurs during mRNA maturation and results in exclusion of intronic sequences and the assembly of consecutive or nonconsecutive exons from pre-mRNA1. The capability to program transcript splicing is highly desirable for synthetic biology and therapeutic applications, for the treatment of monogenic diseases. Since autosomal diseases are often caused by mutations within exons that lead to loss of protein function, removal of the affected exon may provide a therapeutic benefit by enabling translation of truncated protein isoforms free of mutations that are capable of partially fulfilling the physiological role of the full-length protein. Conventional targeted exon skipping has been accomplished by directing antisense oligonucleotides (AONs) to splicing regulatory elements in order to block the native splicing machinery and prevent incorporation of the targeted exon into the mature transcript[5]. The CRISPR–Cas[9] genome editing system has been shown to induce permanent exon skipping[6], which has been harnessed for therapeutic correction of genetic diseases[7]
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