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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.

Large-scale sequencing studies of hematologic malignancies have revealed notable epistasis among high-frequency mutations. One of the most striking examples of epistasis occurs for mutations in RNA splicing factors. These lesions are among the most common alterations in myeloid neoplasms and generally occur in a mutually exclusive manner, a finding attributed to their synthetic lethal interactions and/or convergent effects. Curiously, however, patients with multiple-concomitant splicing factor mutations have been observed, challenging our understanding of one of the most common examples of epistasis in hematologic malignancies. In this study, we performed bulk and single-cell analyses of patients with myeloid malignancy who were harboring ≥2 splicing factor mutations, to understand the frequency and basis for the coexistence of these mutations. Although mutations in splicing factors were strongly mutually exclusive across 4231 patients (q < .001), 0.85% harbored 2 concomitant bona fide splicing factor mutations, ∼50% of which were present in the same individual cells. However, the distribution of mutations in patients with double mutations deviated from that in those with single mutations, with selection against the most common alleles, SF3B1K700E and SRSF2P95H/L/R, and selection for less common alleles, such as SF3B1 non-K700E mutations, rare amino acid substitutions at SRSF2P95, and combined U2AF1S34/Q157 mutations. SF3B1 and SRSF2 alleles enriched in those with double-mutations had reduced effects on RNA splicing and/or binding compared with the most common alleles. Moreover, dual U2AF1 mutations occurred in cis with preservation of the wild-type allele. These data highlight allele-specific differences as critical in regulating the molecular effects of splicing factor mutations as well as their cooccurrences/exclusivities with one another.

Genes encoding the RNA splicing factors SF3B1, SRSF2, and U2AF1 are subject to frequent missense mutations in clonal hematopoiesis and diverse neoplastic diseases. Most "spliceosomal" mutations affect specific hotspot residues, resulting in splicing changes that promote disease pathophysiology. However, a subset of patients carry spliceosomal mutations that affect non-hotspot residues, whose potential functional contributions to disease are unstudied. Here, we undertook a systematic characterization of diverse rare and private spliceosomal mutations to infer their likely disease relevance. We utilized isogenic cell lines and primary patient materials to discover that 11 of 14 studied rare and private mutations in SRSF2 and U2AF1 induced distinct splicing alterations, including partially or completely phenocopying the alterations in exon and splice site recognition induced by hotspot mutations or driving "dual" phenocopies that mimicked two co-occurring hotspot mutations. Our data suggest that many rare and private spliceosomal mutations contribute to disease pathogenesis and illustrate the utility of molecular assays to inform precision medicine by inferring the potential disease relevance of newly discovered mutations.

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.