SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer’s Disease [miSeq]. SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer’s Disease [miSeq]

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 Aβ plaques in Alzheimer’s disease. SPLICER enabled precise and highly efficient exon skipping, which reduced the formation of Aβ42 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: Mammalian cell transfections with Cas9 base editors to monitor DNA editing and RNA expression and alternative splicing to investigate the effect of A>G and C>T SNPs at splice donor and acceptor sites to skip exons. AAV stereotaxic injections into mice brains to moniter DNA editing and exon skipping with base editing tools

外显子跳跃（exon skipping）技术可从成熟mRNA转录本中剔除靶向外显子，在分子与细胞生物学、医学及生物技术领域拥有广泛应用前景。现有外显子跳跃技术涵盖反义寡核苷酸（antisense oligonucleotides）、靶向核酸酶（targetable nucleases）与碱基编辑器（base editors）；尽管此类技术在部分靶向外显子的特定应用中效果良好，但仍存在诸多局限，阻碍其更广泛的落地——例如寡核苷酸类工具存在瞬时作用缺陷，靶向核酸酶类工具受限于PAM基序（PAM motifs）与脱氨酶（deaminases）的序列上下文偏好，而基因编辑工具则易引发非预期的隐蔽剪接（cryptic splicing）。为克服上述局限，我们研发了SPLICER——一款新一代碱基编辑器工具包，由与不同脱氨酶融合的近乎无PAM的Cas9切口酶（near-PAMless Cas9 nickase）变体构成，可同时对剪接受体（splice acceptor, SA）与剪接供体（splice donor, SD）序列进行编辑。同步的SA与SD编辑不仅可提升外显子跳跃效率，还能减少隐蔽剪接、内含子滞留等异常剪接结果。SPLICER可高效编辑外显子剪接位点，涵盖诸多野生型SpCas9碱基编辑器（native SpCas9 BEs）难以实现剪接重编程的外显子。为验证SPLICER的治疗潜力，我们以淀粉样前体蛋白（APP）外显子17为靶向对象——该外显子包含与阿尔茨海默病（Alzheimer’s disease）中β淀粉样蛋白（Aβ）斑块形成相关的氨基酸残基。体外实验中，SPLICER可实现精准且高效的外显子跳跃，减少Aβ42肽的生成；同时在阿尔茨海默病人源化小鼠模型中，该工具可在体内引发DNA编辑与外显子跳跃。综上，SPLICER是一款适用范围广、编辑效率高的外显子跳跃工具包，具备广阔的治疗应用前景。整体实验设计：采用Cas9碱基编辑器转染哺乳动物细胞，以监测DNA编辑、RNA表达及可变剪接情况，探究剪接供体与受体位点处A>G及C>T单核苷酸多态性（single nucleotide polymorphisms, SNPs）对外显子跳跃的影响；通过腺相关病毒（AAV）立体定位注射至小鼠脑部，利用碱基编辑工具监测DNA编辑与外显子跳跃事件。