pgHPRT1.a-c create gDNA excision events that are too small to resolve. Bottom, PCR analysis of the HPRT1 exon two genomic locus. ![]() e, Top, representative Sanger sequencing of pgFARM-edited HPRT1 exon two (gray box). d, Left, RT-PCR analysis of HPRT1 exon two (e2) inclusion. ![]() PgFARM facilitates rapid, programmable exon skipping.Ī, Top, RNA-seq read coverage and sequence conservation across HPRT1 in HeLa/iCas9 cells. However, the vast majority of poison exons have not been functionally interrogated, and their hypothesized essentiality has never been tested. For example, poison exons within splicing factors can mediate gene expression autoregulation 13, 14. Although poison exons do not contribute to the protein-coding capacity of their host genes, a subset are known to play critical cellular roles. Many ultraconserved and highly conserved elements overlap poison exons, defined as alternative exons which interrupt their host genes’ reading frames 13, 14 and trigger nonsense-mediated RNA decay (NMD) 15. The human genome contains 481 “ultraconserved elements” that are perfectly conserved in the mouse and rat genomes 12. “Poison exons” provide a striking example of alternative splicing that is likely critical for organismal function, yet challenging to study. CRISPR/Cas9 has been used to knock out DMD isoforms or long non-coding RNAs by targeting splice sites 10, 11, but has not been applied in a multiplexed fashion for studying alternative isoforms. Antisense oligonucleotides are low-throughput 8, 9, while RNAi does not alter alternative splicing. This disparity between identification and functional characterization of isoforms arises from technological limitations. However, the vast majority of disease-associated RNA isoforms have not been functionally studied, hindering such therapeutic development. Mapping individual mis-spliced isoforms to specific molecular pathologies can enable the rational design of splicing-targeted therapeutics 6, 7. Most biological processes are characterized by alternative splicing 1– 3, which is correspondingly dysregulated in many diseases 4, 5. The essentiality and cancer relevance of poison exons likely contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability. 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. 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. 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 describe pgFARM ( paired guide RNAs for alternative exon re moval), a CRISPR/Cas9-based method to manipulate isoforms independent of gene inactivation. While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms.
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