SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer's Disease [RNA-seq]
Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which has broad applications in molecular and cellular biology, medicine and biotechnology. Existing exon skipping techniques include antisense oligonucleotides, targetable nucleases and base editors, which, while effective for specific applications at some target exons, remain hindered by shortcomings preventing their broader implementation including transient effects in the case of oligonucleotides or limiting PAM motifs, sequence context preferences for deaminases, and undesirable cryptic splicing in the case of gene editing tools. To overcome these limitations, we created SPLICER, a toolbox of next-generation base editors consisting of near-PAMless Cas9 nickase variants fused with different deaminases for simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing not only improves exon skipping rates but also reduces aberrant outcomes such as cryptic splicing and intron retention. SPLICER enables editing of exon splice sites with high efficiency, including many exons refractory to splicing reprogramming by the native SpCas9 BEs. To demonstrate the therapeutic potential of SPLICER, we targeted APP exon 17, which contains the amino acid residues responsible for the formation of Aß plaques in Alzheimer's disease. SPLICER enabled precise and highly efficient exon skipping, which reduced the formation of Aß42 peptides in vitro while inducing DNA editing and exon skipping in vivo within a humanized mouse model of Alzheimer's disease. Overall, SPLICER is a widely applicable and highly efficient toolbox for exon skipping with broad therapeutic applications. Overall design: HEK293T cells were transfected via lipofectamine 2000 with plasmids encoding either a GFP control or SpRY-Cas9-ABE8e to measure the effect that SpRY-Cas9-ABE8e has on both gene expression for the targeted gene (APP exon 17) as well as overall expression on genes associated with Alzheimer's disease. Additionally, overall fold change in gene expression patterns was measured to via DEG to observe if the base editor system had any effects on off-target mRNA expression as well as effects on expression pathways involved with base-excision repair or mismatch DNA repair. Following trnasfection, RNA samples were harvested from cells via RNeasy kit and sequenced.