RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons

Abstract

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 likely contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Isoform cell type specificity in the mouse primary motor cortex

“…The next step after assembling a single-cell isoform atlas as we have done here for the primary motor cortex, is to probe the functional significance of cell type isoform specificity. Recently developed experimental methods for this purpose such as isoforms screens 33 , are a promising direction. This approach is an example of methods that will be key to understanding the significance of the vast isoform diversity in the brain 34 ,…”

Enhancing CRISPR deletion via pharmacological delay of DNA-PK

“…Studies on cultured cells typically report efficiencies in the range 0% -50% of alleles, and often <20% 29,30 , similar to estimates from individual clones 4,17,[24][25][26] . Indeed, a recent publication reported high variation in the efficiencies of paired sgRNA targeting the same region, including many that yielded negligible deletion 30 . Transfection typically yields greater efficiency than viral transduction, possibly due to higher sgRNA levels 32 , but is incompatible with pooled screening.…”

“…We began by replicating CiDER experiments in two widely-used cell lines, HCT116 and HEK293T 30,[58][59][60] (Fig. 2b).…”

“…Homozygous knockout clones may be isolated by screening hundreds of single cells, however this is slow and laborious, and resulting clones may not be representative of the general population 31 . More important than these practical costs, is the potential impact of low deletion rates on the ability to discern bona fide functional effects arising from a given mutation 30 . Non-performing sgRNA pairs are a particular problem for pooled CRISPR-del screens, where they reduce statistical power and lead to false negative results.…”

Androgen signaling connects short isoform production to breakpoint formation at Ewing sarcoma breakpoint region 1 via an R-loop-dependent mechanism

“…Since AR binding at the intron 5 site is more robust in tumors and VCaP cells, we focused on understanding the importance of AR binding to that site by a CRISPR-Cas9 disruption strategy. To avoid the limitations of targeting a short A:T rich sequence, paired guide RNAs (pgRNA) were targeted to sequence flanking the intron 5 ARE ( Figure 2C) as pgRNA targeting is an effective method to achieve targeted deletions (Diao et al, 2017;Gasperini et al, 2017;Thomas et al, 2020). Cells with Cas9 targeted to the intron 5 ARE failed to upregulate ntEWS upon R1881 treatment while not affecting flEWS expression ( Figure 2D).…”