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.
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RNA-seq data generated as part of this study have been deposited in the Gene Expression Omnibus (accession number GSE120703). RNA-seq data generated by TCGA were downloaded from the Cancer Genomics Hub (CGHub) and Genomic Data Commons (GDC). Other data that support this study’s findings are available from the authors upon reasonable request. Source data for Figs. 1–4 and Extended Data Figs. 1, 2, 4, 6 and 10 are presented with the paper.
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We thank M. Gasperini, G. Findlay and J. Shendure for technical assistance and sharing pgRNA constructs, Q. Yan for sharing HeLa/iCas9 cells, A. Geballe for sharing Cas9-expressing IMR90 cells and D. Bennett for sharing Melan-a cells. J.D.T. is a Washington Research Foundation Postdoctoral Fellow. R.K.B. is a Scholar of The Leukemia and Lymphoma Society (1344-18). This research was supported in part by the Edward P. Evans Foundation, NIH/NIDDK (R01 DK103854), NIH/NHLBI (R01 HL128239), NIH/NINDS (P01 NS069539) and the Experimental Histopathology and Genomics Shared Resources of the Fred Hutch/University of Washington Cancer Consortium (P30 CA015704). The results published here are based in part on data generated by The Cancer Genome Atlas Research Network (http://cancergenome.nih.gov).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Sanger sequencing of pgFARM-edited HPRT1 exon two in HeLa/iCas9 cells. b, Long range RT-PCR analysis of HPRT1 exon two skipping. c, RT-PCR analysis of HPRT1 exon two (e2) inclusion before/after Cas9 induction (day 0/day 10) and one week treatment with 6-thioguanine ( + 6TG). d, HPRT1 western blot analysis (n = 1 independent experiments) before (-) and after ( + ) one week treatment with 6TG. e, Cas9-expressing HEK293T cells (n = 3 biological replicates) that were untreated (wild-type) or expressing the indicated pgRNAs followed by one week treatment with 6TG. f, RT-PCR analysis of HPRT1 exon two (e2) inclusion in Cas9-expressing HEK293T cells (n = 3 biological replicates). g, Top, RT-PCR analysis of MET exon 14 (e14) inclusion with ( + ) or without (-) Cas9 expression. Bottom, quantification. (n = 1 independent experiments). h, As for (b), but for MET exon 14. Gray, non-targeting pgRNA; green, pgRNA targeting MET exon 14. See Source Data for uncropped gels. Source data
a, Sanger sequencing of pgFARM-edited MBNL1 exon two in HeLa/iCas9 cells. b, Long range RT-PCR analysis of MBNL1 exon two skipping (n = 1 independent experiments). c, Left, RT-PCR analysis (n = 3 biological replicates per group) of MBNL1 exon five (e5) inclusion in Cas9-expressing IMR90 cells expressing a non-targeting pgRNA (pgNTC) or pgMBNL1.a. Right, quantification of MBNL1 exon 5 inclusion. d, Left and center, RT-PCR analysis and associated quantification of Mbnl1 exon five (e5) inclusion in Cas9-expressing B16-F10 cells expressing the indicated pgRNA. Right, RT-PCR analysis (n = 3 biological replicates per group) and associated quantification of Mbnl1 exon (e5) inclusion in Cas9-expressing Melan-A cells expressing the indicated pgRNA. e, Individual Mbnl1 alleles that were cloned from gDNA of Cas9-expressing B16-F10 cells following delivery of a Mbnl1 exon five-targeting pgRNA and subjected to Sanger sequencing. f, Quantification of total MBNL1 protein levels (top) and MBNL1 protein encoded by the exon five-including isoform (bottom) before (day 0) and after (day 14) Cas9 induction in HeLa/iCas9 cells expressing the indicated pgRNA, measured by immunoblot in Fig. 1l. *, pgRNAs that induced the greatest MBNL1 exon five exclusion. Data are representative of n = 2 independent experiments. g, Scatter plot comparing pgRNA-mediated exclusion of MBNL1 exon five (e5) and inclusion of MBNL2 exon five (e5), a paralogous exon that is regulated by nuclear MBNL1. Datapoints (n = 24) are from HeLa/iCas9 cells treated with pgMBNL1.a, pgMBNL1.d, or pgMBNL1.e pgRNAs for two weeks. r, Pearson correlation; p, associated p-value computed using a two-sided Student’s t-test; shaded region, 95% confidence interval. See Source Data for uncropped gels. Source data
a, As Fig. 2c, but for all TCGA cohorts analyzed in Fig. 2d. p computed with two-sided Mann-Whitney U test. Hinges, notches, and whiskers indicate 25th/75th percentiles, 95% confidence interval, and most extreme datapoints within 1.5X interquartile range from hinge. Sample sizes are BLCA: n = 338; BRCA: n = 1089; COAD: n = 451; ESCA: n = 180; HNSC: n = 40; KICH: n = 62; KIRC: n = 430; KIRP: n = 262; LIHC: n = 350; LUAD: n = 502; LUSC: n = 447; PRAD: n = 481; STAD: n = 30; THCA: n = 362. b, Overall survival of lung adenocarcinoma (LUAD) patients, where patients were stratified according to the relative inclusion of the SMNDC1 poison exon. High poison exon, top tercile of samples; low poison exon, bottom tercile of samples. p computed with a two-sided logrank test. n = 237 (low) and 132 (high) samples. The uneven sample allocation arises from edge effects at the boundaries of terciles (MISO only estimates exon inclusion to two significant digits). c, As (b), but for SMNDC1 gene expression. High expression, top tercile of samples; low expression, bottom tercile of samples. p computed with a two-sided logrank test. n = 169 (low) and 174 (high) samples.
a, Sanger sequencing of pgFARM-edited SMNDC1 poison exon in HeLa/iCas9 cells. Annotations of eliminated (X) or disrupted (↓) sequence elements are indicated. b, Western blot for Cas9 and ACTB in parental PC9 and PC9-Cas9 (n = 3 biological replicates) transgenic cell lines. c, Left, PC9-Cas9 cells expressing the indicated pgRNAs following treatment with 6TG for one week. Right, quantification of cell survival. d, Representative SMNDC1 allele (n = 25 total sequenced alleles) of a PC9-Cas9 clonal cell line isolated following delivery of an SMNDC1 poison exon-targeting pgRNA. e, MaxEnt 3′ splice site scores for unedited (wild-type) or edited SMNDC1 alleles from individual PC9-Cas9 clones. “small” and “medium” indicate alleles containing indels of length ~1–10 bp and > 10 bp without intervening gDNA excision; “gDNA excision” indicates alleles with complete excision of intervening gDNA. Each class of editing event can effectively reduce 3′ splice site strength. f, As Fig. 2j, but restricted to introns that are not NMD-targets (NMD-irrelevant). g, As Fig. 2k, but restricted to introns that are not NMD-targets (NMD-irrelevant). See Source Data for uncropped gels. Source data
a, Regions used to classify each poison exon (n = 12,653) according to its sequence conservation. b, Median conservation scores for each indicated region (violin plot width represents probability density of data distribution). c, Median per-nucleotide sequence conservation for exon groups described in the text. d, Per-nucleotide sequence conservation for an SRSF3 ultraconserved poison exon. e, As (d), but for an MTX2 poorly conserved poison exon. f, The most significant biological processes associated with genes containing unconserved poison exons (n = 2,363), conserved poison exons (n = 352), or conserved non-poison exons (n = 888) (related to Fig. 3c). FDR computed using the Wallenius method and corrected using the Benjamini-Hochberg method. g, pgRNA library summary. h, On-target scores (MIT score) for all gRNAs targeting 3′ splice sites analyzed in our study (“false”) and those included in the final library (“true”). i, As (h), but for off-target scores identified using Cas-OFFinder.
a, pgRNA library generation for Illumina sequencing. b, pgRNA counts throughout the time course (n = 1,000; 3,604; 4,099; 805 for groups, left to right). c, Relative proliferation of HeLa/iCas9 cells expressing an SMNDC1 upstream constitutive exon-targeting pgRNA relative to control pgRNA (non-essential gene CSPG4; n = 2 independent experiments). d, Unnormalized fold-changes for non-targeting pgRNAs (n = 1,000) and pgRNAs targeting unexpressed ( < 1 transcripts per million, TPM) genes, located in genomic regions with the indicated copy numbers (n = 2, 38, 45, and 11, left to right). e, Normalized fold-changes for all non-targeting pgRNAs (NTC; n = 1,000) and pgRNAs targeting the indicated exons (n = 9 pgRNA per exon) in SNRNP70. f, Relative proliferation of HeLa/iCas9 cells expressing a SNRNP70 upstream constitutive exon-targeting pgRNA without (-) or with ( + ) simultaneous overexpression of a SNRNP70-encoding cDNA (n = 6 replicates per condition). g, Representative Sanger sequencing of a pgFARM-edited SNRNP70 upstream exon in HeLa/iCas9 cells (n = 19 total sequenced alleles). h, RNA-seq read coverage across the SNRNP70 locus containing the targeted upstream constitutive exon (gray box) from HeLa/iCas9 cells expressing the indicated pgRNA (n = 1 per pgRNA). Ψ, percent spliced in. i, SNRNP70 poison exon inclusion for HeLa/iCas9 cells expressing the indicated pgRNA relative to a non-targeting pgRNA (n = 1 per pgRNA). j, Scatter plot comparing cassette exon inclusion in HeLa/iCas9 cells treated with a non-targeting control pgRNA (pgNTC) or SNRNP70 upstream constitutive exon-targeting pgRNA (pgSNRNP70). Points are shaded by statistical significance (two-sided Mann-Whitney test). k, As (j), but comparing alternative 5′ splice site usage. For box plots, the line, hinges, and whiskers represent median, 25th and 75th percentiles, and most extreme datapoints within 1.5X interquartile range from hinge. See Source Data for uncropped gels. Source data
a, Normalized pgRNA fold-changes (n = 1,000 and 9 for non- and exon-targeting pgRNAs, respectively). The center line, hinges, and whiskers represent median, 25th and 75th percentiles, and most extreme datapoints within 1.5X interquartile range from hinge. b, RNA-seq read coverage across the SRSF3 locus containing the targeted upstream constitutive exon (gray box) from HeLa/iCas9 cells expressing the indicated pgRNA (n = 1 per pgRNA). Ψ, percent spliced in. c, SRSF3 poison exon inclusion for HeLa/iCas9 cells expressing the indicated pgRNA relative to a non-targeting pgRNA (n = 1 per pgRNA). d, SRSF3 RNA binding motif enrichment in differentially spliced exons (n = 2,046 left; 727 right) in HeLa/iCas9 cells expressing the indicated pgRNA. Data presented as mean ± 95% confidence interval computed by bootstrapping. e, Scatter plot comparing cassette exon inclusion in HeLa/iCas9 cells treated with a non-targeting control pgRNA (pgNTC) or AAVS1-targeting control pgRNA (pgAAVS1). Points are shaded by statistical significance (two-sided Mann-Whitney U test). f, RNA-seq read coverage across the entire SNRNP70 locus in HeLa/iCas9 cells expressing the indicated pgRNA (n = 1 per pgRNA). g, As (f), but for SRSF3 (n = 1 per pgRNA).
a, HeLa/iCas9 cells (n = 4 biological replicates) treated with the poison exon pgRNA library and grown in the presence ( + dox) or absence (- dox) of active Cas9. b, Scatter plots comparing normalized fold-changes (day 14 vs. day 0; n = 963 targeted exons) estimated with each replicate of the cell viability screen in HeLa/iCas9 cells. Pearson correlations for individual replicate comparisons are indicated. c, Normalized fold-changes for pgRNAs targeting exons in unexpressed (TPM ≤ 1; n = 96 for HeLa/iCas9 and 128 for PC9-Cas9) or highly expressed (TPM ≥ 10; n = 681 for HeLa/iCas9 and 661 for PC9-Cas9) genes. Each dot represents the median fold-change computed over all pgRNAs targeting exons in the indicated groups for a representative replicate from the screens in HeLa/iCas9 (left; n = 5) and PC9-Cas9 (right; n = 4) cells. TPM, transcripts per million. d, Normalized fold-changes for pgRNAs targeting lowly expressed genes (TPM < 5) located in genomic regions with the indicated copy numbers (n = 6, 165, and 14 per group, left to right, for HeLa/iCas9; n = 60, 107, and 45 per group, left to right, for PC9-Cas9). e, Rank plot of mean normalized fold-changes for conserved poison (orange) or upstream constitutive exons (purple) based on all replicates of the HeLa/iCas9 viability screen. f, As (e), but for all replicates of the PC9-Cas9 viability screen. For box plots, the center line, hinges, and whiskers represent median, 25th and 75th percentiles, and most extreme datapoints within 1.5X interquartile range from hinges, respectively.
a, Sanger sequencing of pgFARM-edited CPSF4 poison exon in HeLa/iCas9 cells. Annotations of eliminated (X) or disrupted (↓) sequence elements are indicated. b, RNA-seq read coverage across the entire CPSF4 locus in HeLa/iCas9 cells expressing a CPSF4 poison exon-targeting pgRNA (pgCPSF4; n = 1). We observed no read coverage indicative of cryptic splicing in pgCPSF4-treated cells. The two sets of splice junction reads downstream of the CPSF4 poison exon correspond to usage of endogenous (naturally occurring in unedited cells) competing 3′ splice sites. c, As (b), but for an SMG1 poison exon-targeting pgRNA (pgSMG1; n = 1). d, Scatter plot comparing normalized fold-changes for pgRNAs targeting a poison exon compared to matched upstream coding exon within the same gene.
a, Tumors derived from parental PC9 or PC9-Cas9 cells (n = 4 per group). b, Mice from early and late tumor time points (n = 4 and 10 tumors, respectively). c, pgRNA Illumina libraries. d, Pearson correlation (r) matrix for xenograft screen samples. Unsupervised clustering of library depth-normalized pgRNA counts by the complete-linkage method. e, Normalized counts (mean ± S.D.) for gRNAs targeting coding exons in the indicated genes. Data from Chen et al, 2015 (n = 1, 6, 3, and 9 for groups, left to right). f, Relative cell number (mean ± S.D.) for PC9-Cas9 cells expressing a pgRNA targeting the indicating exons (n = 3 per group). g, Progression-free survival of lung adenocarcinoma patients (n = 167/171 for low/high categories), where patients were stratified by inclusion of tumor-suppressive poison exons. h, As (g), but for overall survival. i, As (g), but for essential poison exons (n = 166/169 for low/high categories). j, As (i), but for overall survival. See Source Data for uncropped gels. Source data
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Thomas, J.D., Polaski, J.T., Feng, Q. et al. RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons. Nat Genet 52, 84–94 (2020) doi:10.1038/s41588-019-0555-z