RNA targeting with CRISPR Cas13a

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Cloning of orthologues for activity screen and recombinant expression

We synthesized human codon-optimized versions of 15 CRIPSR Cas13a orthologues (Genscript) and cloned them into pACYC184 under a pLac promoter. Adjacent to the Cas13a expression cassette, we cloned the orthologue’s corresponding direct repeats flanking either a β-lactamase-targeting or non-targeting spacer. Spacer array expression was driven by the J23119 promoter.

For purification of LwaCas13a, we cloned the mammalian codon-optimized LwaCas13a sequence into a bacterial expression vector for protein purification (6× His/Twin Strep SUMO, a pET-based expression vector received as a gift from I. Finkelstein, University of Texas-Austin).

Bacterial in vivo testing for Cas13a activity and PFS identity

In brief, CRIPSR Cas13a was programmed to target a 5′ stretch of sequence on the β-lactamase transcript flanked by randomized PFS nucleotides. Cas13a cleavage activity resulted in death of bacteria under ampicillin selection, and PFS depletion was subsequently analysed by next-generation sequencing.

To test for activity of CRIPSR Cas13a orthologues, 90 ng of orthologue expression plasmid with either targeting or non-targeting guide was co-transformed with 25 ng of a previously described β-lactamase target plasmid8 into NovaBlue Singles Competent Cells (Millipore). Post-transformation, cells were diluted, plated on LB-agar supplemented with 100 μg μl−1 ampicillin and 25 μg μl−1 chloramphenicol, and incubated at 37 °C overnight. Transformants were counted the next day.

For determination of LshCas13a and LwaCas13a PFS identity, 40 ng of orthologue expression plasmid with either targeting or non-targeting spacer was co-transformed with 25 ng of β-lactamase target plasmid into two aliquots of NovaBlue GigaSingles (Millipore) per biological replicate. Two biological replicates were performed. Post-transformation, cells were recovered at 37 °C in 500 μl of SOC (ThermoFisher Scientific) per biological replicate for 1 h, plated on bio-assay plates (Corning) with LB-agar (Affymetrix) supplemented with 100 μg μl−1 ampicillin and 25 μg μl−1 chloramphenicol, and incubated at 37 °C for 16 h. Colonies were then harvested by scraping, and plasmid DNA was purified with NuceloBond Xtra EF (Macherey-Nagel) for subsequent sequencing.

Harvested plasmid samples were prepared for next-generation sequencing by PCR with barcoding primers and Illumina flow cell handles using NEBNext High Fidelity 2X Master Mix (New England Biosciences). PCR products were pooled and gel extracted using a Zymoclean gel extraction kit (Zymo Research) and sequenced using a MiSeq next-generation sequencing machine (Illumina).

Computational analysis of PFS

From next-generation sequencing of the LshCas13a and LwaCas13a PFS screening libraries, we aligned the sequences flanking the randomized PFS region and extracted the PFS identities. We collapsed PFS identities to four nucleotides to improve sequence coverage, counted the frequency of each unique PFS, and normalized to total read count for each library with a pseudocount of 1. Enrichment of each distribution was calculated against the pACYC184 control (no protein/guide locus) as −log₂(f_condition/f_pACYC184), where fcondition is the frequency of PFS identities in the experimental condition and f_pACYC184 is the frequency of PFS identities in the pACYC184 control. To analyse a conserved PFS motif, top depleted PFS identities were calculated using each condition’s non-targeting control as follows: −log₂(f_i,targeting/f_{i,non-targeting}) where f_i,targeting is the frequency of PFS identities in condition i with targeting spacer and fi,non-targeting is the frequency of PFS identities in condition i with non-targeting spacer. PFS motifs were analysed for a range of thresholds.

Purification of CRIPSR Cas13a

Purification of LwaCas13a was performed as previously described9. In brief, CRIPSR LwaCas13a bacterial expression vectors were transformed into Rosetta 2(DE3)pLysS singles Competent Cells (Millipore) and 4 l of Terrific Broth 4 growth media were seeded with a starter culture. Cell protein expression was induced with IPTG and after overnight growth, the cell pellet was harvested and stored at −80 °C. Following cell lysis, protein was bound using a StrepTactin Sepharose resin (GE) and protein was eluted by SUMO protease digestion (ThermoFisher). Protein was further purified by cation exchange using a HiTrap SP HP cation exchange column (GE Healthcare Life Sciences) and subsequently by gel filtration using a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences), both steps via FPLC (AKTA PURE, GE Healthcare Life Sciences). Final fractions containing LwaCas13a protein were pooled and concentrated into Storage Buffer (600 mM NaCl, 50 mM Tris-HCl pH 7.5, 5% glycerol, 2 mM DTT) and aliquots were frozen at −80 °C for long-term storage.

Cloning of mammalian and plant expression constructs

The human codon-optimized CRIPSR Cas13a gene was synthesized (Genscript) and cloned into a mammalian expression vector with either a nuclear export sequence or NLS under expression of the EF1-a promoter. Because of the stability conferred by monomeric-super-folded GFP (msfGFP), we fused msfGFP to the C terminus of LwaCas13a. The full-length direct repeat of LwaCas13a was used for cloning the guide backbone plasmid with expression under a U6 promoter. The catalytically inactive LwaCas13a–msfGFP construct (dead LwaCas13a or dLwaCas13a) was generated by introducing R474A and R1046A mutations in the two HEPN domains. A drug-selectable version of LwaCas13a–msfGFP was generated by cloning the protein into a backbone with the Blasticidin selection marker linked to the C terminus via a 2A peptide sequence. The negative-feedback version of the dLwaCas13a–msfGFP construct (dLwaCas13a–NF) was generated by cloning a zinc-finger binding site upstream of the promoter of dLwaCas13a–msfGFP and fusing a zinc finger and KRAB domain to the C terminus.

The reporter luciferase construct was generated by cloning Cypridinia luciferase (Cluc) under expression of the CMV promoter and Gaussia luciferase (Gluc) under expression of the EF1-a promoter, both on a single vector. Expression of both luciferases on a single vector allowed one luciferase to serve as a dosing control for normalization of knockdown of the other luciferase, controlling for variation due to transfection conditions.

For the endogenous knockdown experiments in Fig. 1g, guides and shRNAs were designed using the RNAxs siRNA design algorithm. The prediction tool was used to design shRNAs, and guides were designed in the same location to allow for comparison between shRNA and LwaCas13a knockdown.

For the plant knockdown experiments, the rice actin promoter (pOsActin) was PCR amplified from pANIC6A and LwaCas13a was PCR amplified from human expression LwaCas13a constructs. These fragments were ligated into existing plant expression plasmids such that LwaCas13a was driven by the rice actin promoter and transcription was terminated by the HSP terminator while the LwaCas13a gRNAs were expressed from the rice U6 promoter (pOsU6).

Protoplast preparation

Green rice protoplasts (O. sativa L. ssp. japonica var. Nipponbare) were prepared as previously described with slight modifications. Seedlings were grown for 14 days and protoplasts were resuspended in mMG buffer containing 0.1 M CaCl₂. This modified mMG buffer was used to prepare fresh 40% PEG buffer as well as in place of WI buffer. Finally, protoplasts were kept in total darkness for 48 h post-transformation. All other conditions were as previously described.

Nucleic-acid target and crRNA preparation for in vitro reactions and collateral activity assays

To generate nucleic-acid targets, oligonucleotides were PCR amplified with KAPA Hifi Hot Start (Kapa Biosystems). dsDNA amplicons were gel extracted and purified using a MinElute gel extraction kit (Qiagen). The resulting purified dsDNA was transcribed via overnight incubation at 30 °C with a HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs). Transcribed RNA was purified using a MEGAclear Transcription Clean-up kit (Thermo Fisher).

To generate crRNAs, oligonucleotides were ordered as DNA (Integrated DNA Technologies) with an additional 5′ T7 promoter sequence. crRNA template DNA was annealed with a T7 primer (final concentrations 10 μM) and transcribed via overnight incubation at 37 °C with a HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs). The resulting transcribed crRNAs were purified with RNAXP clean beads (Beckman Coulter), using a 2× ratio of beads to reaction volume, supplemented with additional 1.8× ratio of isopropanol (Sigma).

CRIPSR LwaCas13a cleavage and collateral activity detection

For biochemical characterization of CRIPSR LwaCas13a, assays were performed as previously described. In brief, nuclease assays were performed with 160 nM of end-labelled single-stranded (ss)RNA target, 200 nM purified LwaCas13a, and 100 nM crRNA, unless otherwise indicated. All assays were performed in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl₂, pH 7.3). For array processing, 100 ng of in vitro transcribed array was used per nuclease assay. Reactions were allowed to proceed for 1 h at 37 °C (unless otherwise indicated) and were then quenched with proteinase buffer (150 U ml−1 proteinase K, 60 mM EDTA, and 4 M urea) for 15 min at 37 °C. The reactions were then denatured with 4.5 M urea denaturing buffer at 95 °C for 5 min. Samples were analysed by denaturing gel electrophoresis on 10% PAGE TBE-Urea (Invitrogen) run at 45 °C. Gels were imaged using an Odyssey scanner (LI-COR Biosciences).

Collateral activity detection assays were performed as previously described. In brief, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNase Alert v2, Thermo Scientific), 2 μl mouse RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic-acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl₂, pH 7.3). Reactions were allowed to proceed for 1–3 h at 37 °C (unless otherwise indicated) on a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min.

Cloning of tiling guide screens

For tiling guide screens, spacers were designed to target mRNA transcripts at even intervals to fully cover the entire length of the transcript. Spacers (ordered from IDT) were annealed and golden-gate cloned into LwaCas13a guide expression constructs with either a tRNAval promoter (Gluc and Cluc screens) or U6 promoter (all endogenous screens).

Mammalian cell culture and transfection for knockdown with CRIPSR/Cas13a

All mammalian cell experiments were performed in the HEK293FT line (American Type Culture Collection (ATCC)) unless otherwise noted. HEK293FT cells were cultured in Dulbecco’s Modified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (VWR Seradigm) and 1× penicillin–streptomycin (Thermo Fisher Scientific). Cells were passaged to maintain confluency below 70%. For experiments involving A375 (ATCC), cells were cultured in RPMI Medium 1640 (Thermo Fisher Scientific) supplemented with 9% fetal bovine serum (VWR Seradigm) and 1× penicillin–streptomycin (Thermo Fisher Scientific). The HEK293FT cells were not authenticated after receiving them from ATCC and were not checked for mycoplasma contamination.

To test knockdown of endogenous genes, Lipofectamine 2000 (Thermo Fisher Scientific) transfections were performed with 150 ng of LwaCas13a plasmid and 250 ng of guide plasmid per well, unless otherwise noted. Experiments testing knockdown of reporter plasmids were supplemented with 12.5 ng reporter construct per well. Sixteen hours before transfection, cells were plated in 96-well plates at approximately 20,000 cells per well and allowed to grow to 90% confluency overnight. For each well, plasmids were combined with Opti-MEM I Reduced Serum Medium (Thermo Fisher) to a total of 25 μl, and separately 0.5 μl of Lipofectamine 2000 was combined with 24.5 μl of Opti-MEM. Plasmid and Lipofectamine solutions were then combined, incubated for 5 min, and slowly pipetted onto cells to prevent disruption.

Transformation of green rice protoplasts

For the green rice experiments, plasmids expressing each CRIPSR LwaCas13a and the corresponding guide RNA were mixed in equimolar ratios such that a total of 30 μg of DNA was used to transform a total of 200,000 protoplasts per transformation.

Measurement of luciferase activity

Media containing secreted luciferase was harvested 48 h after transfection, unless otherwise noted. Media were diluted 1:5 in PBS and then luciferase activity was measured using BioLux Cypridinia and Biolux Gaussia luciferase assay kits (New England Biolabs) on a Biotek Synergy 4 plate reader with an injection protocol. All replicates were performed as biological replicates.

Harvest of total RNA and quantitative PCR

For gene expression experiments in mammalian cells, cell harvesting and reverse transcription for cDNA generation was performed using a previously described modification of the commercial Cells-to-Ct kit (Thermo Fisher Scientific) 48 h after transfection. Transcript expression was then quantified with qPCR using Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan qPCR probes (Thermo Fisher Scientific) with GAPDH control probes (Thermo Fisher Scientific). All qPCR reactions were performed in 5-μl reactions with four technical replicates in a 384-well format, and read out using a LightCycler 480 Instrument II (Roche). For multiplexed targeting reactions, readout of different targets was performed in separate wells. Expression levels were calculated by subtracting housekeeping control (GAPDH) cycle threshold (Ct) values from target Ct values to normalize for total input, resulting in ΔCt levels. Relative transcript abundance was computed as 2−ΔCt. All replicates were performed as biological replicates.

For gene expression experiments in plant cells, total RNA was isolated after 48 h of incubation using Trizol according to the manufacturer’s protocol. One nanogram of total RNA was used in a SuperScript III Plantinum SYBR Green One-Step qRT–PCR Kit (Invitrogen) according to the manufacturer’s protocol. All samples were run in technical triplicate of three biological replicates in a 384-well format on a LightCycler 480 Instrument (Roche). All PCR primers were verified as being specific on the basis of melting curve analysis and were as follows: OsEPSPS (Os06g04280), 5′-TTGCCATGACCCTTGCCGTTGTTG-3′ and 5′-TGATGATGCAGTAGTCAGGACCTT-3′; OsHCT (Os11g07960), 5′-CAAGTTTGTGTACCCGAGGATTTG-3′ and 5′-AGCTAGTCCCAATAAATATGCGCT-3′; OsEF1a (Os03g08020), 5′-CTGTAGTCGTTGGCTGTGGT-3′ and 5′-CAGCGTTCCCCAAGAAGAGT-3′. Primers for OsEF1a were previously described.

For analysis of RNA quality post-knockdown with CRIPSR LwaCas13a, total RNA was harvested by lysing cells using TRI Reagent and purifying the RNA using the Direct-zol RNA MiniPrep Plus kit (Zymo). Four nanograms of total RNA were analysed using a RNA 6000 Pico Bioanalyzer kit (Agilent).

Computational analysis of target accessibility

To first analyse target accessibility, top guides from the tiling screen were analysed to determine whether they grouped closer together than expected under the assumption that if there were regions of accessibility, multiple guides in that region would be expected to be highly active. Top guides were defined as the top 20% of performing guides for the Gluc tiling screen and top 30% of performing guides for the Cluc, KRAS, and PPIB tiling screens. A null probability distribution was generated for pairwise distances between guides by randomly simulating 10,000 guide positions and then comparing with experimentally determined top guide pairwise distances.

Accessibility was predicted using the RNApl fold algorithm in the Vienna RNA software suite25. The default window size of 70 nt was used and the probability of a target region being unpaired was calculated as the average of the 28 single-nucleotide unpaired probabilities across the target region. These accessibility curves were smoothed and compared with smoothed knockdown curves across each of the four transcripts, and correlations between the two factors and their significance were computed using Pearson’s correlation coefficient with the SciPy Python package (pearsonr function). The probability space of these two factors was also visualized by performing two-dimensional kernel density estimation across the two variables.

RNA sequencing and analysis

For specificity analysis of CRIPSR LwaCas13a knockdown, RNA-seq was performed on mRNA from knockdown experiments involving both LwaCas13a and shRNA constructs. Total RNA was prepared from transfection experiments after 48 h using a Qiagen RNeasy Plus Mini kit. mRNA was then extracted using a NEBNext Poly(A) mRNA Magnetic Isolation Module and RNA-seq libraries were prepared using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina. RNA-seq libraries were sequenced on an Illumina NextSeq instrument with at least 10 million reads per library.

An index was generated using the RefSeq GRCh38 assembly and reads were aligned and quantified using Bowtie and RSEM version 1.2.31 with default parameters26. Transcripts per million (TPM) values were used for expression counts and were transformed to log-space by taking the log₂(TPM + 1).

To find differentially expressed genes, Student’s t-test was performed on the three targeting replicates versus the three non-targeting replicates. The statistical analysis was only performed on genes that had a log₂(TPM + 1) value greater than 2.5 in at least two of the six replicates. Only genes that had a differential expression greater than 2 or less than 0.75 and a false discovery rate <0.10 were reported to be significantly differentially expressed.

Cross-correlations between replicates and averages of replicates were performed using Kendall’s tau coefficient. The variation of shRNA versus LwaCas13a libraries was analysed by considering the distribution of standard deviations for gene expression across the six replicates (three targeting and three non-targeting replicates) and represented as violin plots.

Cell viability assay

Mammalian cells were transfected with luciferase reporter target, guide plasmid, and either CRIPSR LwaCas13a or drug-selectable LwaCas13a. Twenty-four hours after transfection, cells were split 1:5 into fresh media and drug-selectable LwaCas13a samples were supplemented with 10 μg ml−1 Blasticidin S (Thermo Fisher Scientific). After 48 h of additional growth, cells were assayed for luciferase knockdown, maintenance of LwaCas13a expression via GFP fluorescence measurement on a multimode plate reader (Biotek Neo2), and cell growth by CellTiter-Glo Luminescent Cell Viability Assay (Promega).

Quantifying dLwaCas13a binding with RNA immunoprecipitation

For RNA immunoprecipitation experiments, HEK293FT cells were plated in six-well plates and transfected with 1.3 μg of dLwaCas13a expression plasmid and 1.7 μg of guide plasmid, with an additional 150 ng of reporter plasmid for conditions involving reporter targeting. Forty-eight hours after transfection, cells were washed twice with ice-cold PBS (Sigma) and fixed with 0.2% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min at room temperature. After fixation, the paraformaldehyde was removed, 125 mM glycine in PBS was added to quench crosslinking, and the cells were incubated for 10 min. Cells were washed twice with ice-cold PBS, harvested by scraping, and the cell suspension was centrifuged at 800g for 4 min to pellet the cells. The supernatant was removed and the pellet was washed with PBS before lysis. Cells were lysed with 200 μl of 1× RIPA Buffer (Cell Signaling) supplemented with cOmplete ULTRA Tablets, EDTA-free (Sigma) and ribonuclease inhibitor (Sigma R1158). Cells were allowed to lyse on ice for 10 min and then sonicated for 2 min with a 30 s on/30 s off cycle at low intensity on a Bioruptor sonicator (Diagenode). Insoluble material was pelleted by centrifugation at 16,000g for 10 min at 4 °C, and the supernatant containing cleared lysate was used for pulldown with magnetic beads.

To conjugate antibodies to magnetic beads, 100 μl per sample of Dynabeads Protein A for Immunoprecipitation (Thermo Fisher Scientific) were pelleted by application of a magnet, and the supernatant was removed. Beads were resuspended in 200 μl of wash buffer (PBS supplemented with 0.02% Tween 20 (Sigma)) and 5 μg of rabbit anti-Mouse IgG (Sigma M7023) was added. The sample was incubated for 10 min at room temperature on a rotator to allow antibody to conjugate to the beads. After incubation, beads were pelleted using a magnet, supernatant was removed, and beads were washed twice with wash buffer. The pellet was resuspended in 100 μl wash buffer and split into two 50 μl volumes for conjugation of anti-HA antibody (Thermo Fisher Scientific 26183) or IgG antibody control (Sigma I5381). For each antibody, 2.5 μg of antibody was added with 200 μl wash buffer and incubated for 10 min at room temperature on a rotator. After incubation, beads were pelleted using a magnet and washed twice with wash buffer, and resuspended in 200 μl 1× RIPA with ribonuclease inhibitor (Sigma R1158) and protease inhibitor cocktail (Sigma P8340). One hundred microlitres of sample lysate were added to beads and rotated overnight at 4 °C.

After incubation with sample lysate, beads were pelleted, washed three times with 1× RIPA, 0.02% Tween 20, and then washed with DNase buffer (350 mM Tris-HCl (pH 6.5); 50 mM MgCl₂; 5 mM DTT). Beads were resuspended in DNase buffer and TURBO DNase (Life Technologies) was added to a final concentration of 0.08 units per microlitre. DNase was incubated for 30 min at 37 °C on a rotator. Proteins were then digested by addition of Proteinase K (New England Biosciences) to a final concentration of 0.1 units per microlitre and incubated at 37 °C with rotation for an additional 30 min. For denaturation and purification, urea (Sigma) was added to a final concentration of 2.5 M, samples were incubated for 30 min, and RNA was purified using a Direct-Zol RNA miniprep (Zymo Research). Purified RNA was reverse transcribed to cDNA using the qScript Flex cDNA (Quantabio) and pulldown was quantified with qPCR using Fast Advanced Master Mix and TaqMan qPCR probes. All qPCR reactions were performed in 5-μl reactions with 4 technical replicates in 384-well format, and read out using a LightCycler 480 Instrument II. Enrichment was quantified for samples compared with their matched IgG antibody controls.

Translocation measurement of CRIPSR LwaCas13a and LwaCas13a-NF

HEK293FT cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips (Corning) and transfected with 150 ng dLwaCas13a–NF vector and 300 ng guides for imaging ACTB. For translocation experiments, cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100 after 48 h, and mounted using anti-fade mounting medium with DAPI (Vectashield). Confocal microscopy was performed with a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.

Nuclear export of dLwaCas13a-NF with guides targeting ACTB mRNA was analysed by measuring the average cytoplasmic and nuclear msfGFP fluorescence and comparing the ratio across many cells between targeting and non-targeting conditions.

FISH of ACTB transcript

HEK293FT cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips (Corning) and transfected with 75 ng dLwaCas13a–NF vector and 250 ng guides for imaging ACTB. After 48 h, cells were fixed with 4% PFA for 45 min. A QuantiGene viewRNA ISH Cell assay kit (Affymetrix) was used for performing FISH on the cell samples according to the manufacturer’s protocol. After finishing the FISH procedure, coverslips were mounted using anti-fade mounting medium (Vectashield). Confocal microscopy was performed using a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.

Tracking of LwaCas13a to stress granules

HEK293FT cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips (Corning) and transfected with 75 ng dLwaCas13a-NF vector and 250 ng guides for imaging ACTB. For stress granule experiments, 200 μM sodium arsenite was applied for 1 h before fixing and permeabilizing the cells. For immunofluorescence of G3BP1, cells were blocked with 20% goat serum, and incubated overnight at room temperature with anti-G3BP1 primary antibody (Abnova H00010146-B01P). Cells were then incubated for 1 h with secondary antibody labelled with Alexa Fluor 594 and mounted using anti-fade mounting medium with DAPI (Vectashield). Confocal microscopy was performed using a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.

Stress granule co-localization with dLwaCas13a–NF was calculated using the average msfGFP and G3BP1 signal per cell using Pearson’s correlation coefficient. The co-localization analyses were performed in the image analysis software FIJI27 using the Coloc 2 plugin.

For live imaging experiments, HEK293FT cells were plated in 96-well tissue culture plates and transfected with 150 ng dLwaCas13a–NF vector, 300 ng guides for imaging ACTB, and 5 ng of G3BP1–RFP reporter. After 48 h, the cells were subjected to 0 μM or 400 μM sodium arsenite and imaged every 15 min for 2 h on an Opera Phenix High Content Screening System (PerkinElmer) using the spinning disk confocal setting with a 20× water objective. Cells were maintained at 37 °C in a humidified chamber with 50% CO₂. Live-cell dLwaCas13a–NF co-localization with G3BP1–RFP in stress granules was measured using Opera Phenix Harmony software (PerkinElmer).

Data availability

CRIPSR LwaCas13a (C2c2) expression plasmids are available from Addgene under UBMTA. Patent applications have been filed relating to work in this manuscript. Support forums and computational tools including relevant code for data analysis are available via the Zhang laboratory website (http://www.genome-engineering.org) and Github (https://github.com/fengzhanglab). High-throughput sequencing data related to this study are available at BioProject under accession number PRJNA383832. Raw data represented in main figures in this study are included in this published article and its Supplementary Information. Additional datasets are available from the corresponding author on reasonable request.