期刊
NATURE GENETICS
卷 52, 期 1, 页码 84-+出版社
NATURE PORTFOLIO
DOI: 10.1038/s41588-019-0555-z
关键词
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资金
- Edward P. Evans Foundation
- NIH/NIDDK [R01 DK103854]
- NIH/NHLBI [R01 HL128239]
- NIH/NINDS [P01 NS069539]
- Experimental Histopathology and Genomics Shared Resources of the Fred Hutch/University of Washington Cancer Consortium [P30 CA015704]
While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM (paired guide RNAs for alternative exon removal), a CRISPR-Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention. We generalized this method to a pooled screen to measure the functional relevance of 'poison' cassette exons, which disrupt their host genes' reading frames yet are frequently ultraconserved. Many poison exons were essential for the growth of both cultured cells and lung adenocarcinoma xenografts, while a subset had clinically relevant tumor-suppressor activity. The essentiality and cancer relevance of poison exons are likely to contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability. pgFARM (paired guide RNAs for alternative exon removal) is a CRISPR-Cas9-based approach to manipulate alternative splicing and identify functional roles for individual exons, including poison exons with essential and tumor-suppressor roles.
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