Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we developed and methodically tested an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions in the mature RNA. We performed a high-throughput essentiality screen, quantitative RT-PCR assays, and PacBio long read sequencing to affirm our ability to specifically target and robustly knockdown individual RNA isoforms. Using the example gene RBFOX2, we validated protein-level changes and assessed the functional impact of isoform-specific knockdown. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions annotated in GENCODE for multi-isoform genes and our gRNA efficacy predictions, we estimate that our junction-centric strategy can uniquely target up to 89% of human RNA isoforms, including 50,066 protein-coding and 11,415 lncRNA isoforms. Importantly, this specificity spans all transcriptional and splicing events, including exon skipping and inclusion, alternative 5’ and 3’ splice sites, and alternative starts and ends.

Protein Digestion Whole cell lysate pellets were prepared for each of three conditions: Control, N-short Knockdown, or N-long Knockdown. Approximately 10% of each lysate was used as input for each of two filter-aided sample preparations (FASP) adapted from (Wiśniewski 2018): one using Trypsin (Promega, V5280) and the other using Asp-N (Promega, VA1160), 1 µg. Each digestion yielded approximately 20 µg of peptide as assayed by NanoDrop A280 (ThermoFisher Scientific). Peptides were desalted using Pierce Peptide Desalting Spin Columns (Pierce, ThermoFisher Scientific). The manufacturer’s protocol was used with a substitution of 0.1% trifluoroacetic acid (TFA) with 0.1% formic acid (Optima LC/MS grade, Thermo Fisher Scientific). LC-MS Targeting of RBFOX2 peptides Desalted samples were analyzed in triplicate by nanoLC-MS/MS using a Dionex Ultimate 3000 (Thermo Fisher Scientific, Bremen, Germany) coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Each injection of approximately 1 μg peptides was loaded onto an Acclaim PepMap 100 trap column (300 μm × 5 mm × 5 μm C18) and gradient-eluted from an Aurora Ultimate TS analytical column (75 μm × 25 cm, 1.7 μm C18) equilibrated in 100% solvent A (0.1% formic acid in water) and 0% solvent B (80% acetonitrile in 0.1% formic acid). The peptides were eluted into the mass spectrometer at 400 nL/min up to 100% B over a period of 90 minutes. MS2 spectra were acquired using the tMS2 (otherwise known as PRM) targeting method. Using this method, only the m/z corresponding to peptides of interest were selected for MS2 acquisition. Separate lists were used for Trypsin-generated peptides and AspN-generated peptides (see Supplemental Table S4 for list of peptides). The following instrument settings were used: positive ion mode was used with 1.7 kV at the spray source, RF lens at 30% with advanced peak determination and XCalibur version 4.5.445.18. Full MS scans were acquired in the Orbitrap from 300 to 2000 m/z with 120,000 resolution. In parallel, the tMS2 targeting was performed on a list of m/z corresponding to either 11 tryptic or 18 AspN-derived peptides of interest. Ions were isolated in the quadrupole, HCD was used for fragmentation (30% normalized collision energy). The resulting fragments were detected in the Orbitrap at 15,000 resolution with standard AGC target and dynamic maximum injection time mode. Data analysis using Skyline Target peptide detection was evaluated using the Skyline software suite (MacLean et al. 2010) (Skyline (64-bit) 24.1.0.199 (6a0775ef83)). Two tryptic peptides shared among all RBFOX2 isoforms, GFGFVTFENSADADR and ILDVEIIFNER, met dot product spectral correlation criterion of at least 0.5. For each common peptide, the fragment ion intensity from each triplicate injection was normalized relative to the average fragment ion intensity of the Control.

RBFOX2 knockdown HUES66 cells (3 days after induction with 1 µg/mL doxycycline) were washed with PBS and then lysed with 300ul ice cold RIPA (Thermo #89900) supplemented with 1mM PMSF (Thermo #36978) and 1x protease inhibitor cocktail (Sigma #P8340) in a 12-well plate on ice for 5 minutes. Cells were scraped, transferred to 1.7ml microcentrifuge tubes and sonicated at 20% intensity for 1x 30 second pulse (Qsonica Q125 Sonicator with ⅛” diameter probe). Samples were centrifuged at ~14,000 x g for 15 minutes at 4°C and the supernatant was transferred to a new tube.