Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

从选择性剪接的 mRNA 表达外显子特异性亚型是一种基本机制，可显着扩展细胞的蛋白质组。然而，评估可变剪接的传统方法要么是消耗性的和工作密集型的，要么不在蛋白质水平上纵向量化亚型表达。在这里，我们因此开发了一种外显子特异性异构体表达报告系统 (EXSISERS)，该系统通过高效的内含肽介导的蛋白质从新生多肽链中无痕地切除报告蛋白，从而非侵入性地报告内源基因的外显子异构体的翻译拼接。我们应用 EXSISERS 来量化微管相关蛋白 tau ( MAPT ) 中疾病相关外显子 10 的包含) 在患者来源的诱导多能干细胞中，并筛选了基于 Cas13 的 RNA 靶向效应子的异构体特异性。我们还将细胞存活与 FOXP1 外显子 18b 的包含相结合， FOXP1参与维持胚胎干细胞的多能性，并证实 MBNL1 是外显子 18b 排除的主导因素。EXSISERS 能够以高灵敏度和细胞分辨率对外显子特异性同种型表达进行无中断和多模式监测，并实现外显子特异性治疗干预的高通量筛选。