nes gfp  (Addgene inc)

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: The fusion genes were each cloned into an IRES-eGFP reporter construct, which is subsequently transcribed into a single chimeric RNA encoding a fusion protein, IRES sequence, and eGFP. Psp Cas13b cleaves the chimeric RNA at the fusion gene breakpoint. The RNA fragments lose key RNA features like their 5’cap, 5’/3’UTRs, and polyA tail, and therefore undergo RNA degradation and/or inefficient protein translation.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Clone Assay, Construct, Sequencing

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a-c, g) EGFP reporter assay to assess the silencing efficiency of tiled Psp Cas13b crRNAs with 3-nucleotide resolution targeting the breakpoint region of fusion transcripts BCR::ABL1-p190 (a) , SNX2::ABL1 (b) SFPQ::ABL1 (c) and (g) BCR::ABL1-p210. Data points in the graphs are averages of mean fluorescence from 4 representative fields of view per condition imaged; N = 3. The data are represented in arbitrary units (A.U.). Errors are SEM and p -values of the one-way ANOVA test are indicated (95% confidence interval). (d-f) RT-PCR assays measuring silencing efficiency of tiled Psp Cas13b crRNAs (3-nucleotide resolution) targeting the breakpoint regions of fusion transcripts (d) , BCR::ABL1-p190, (e) SNX2::ABL1 and (f) SFPQ::ABL1; N = 3; Data are normalised means and errors are SEM; Results are analysed by one-way ANOVA test with p -values indicated (95% confidence interval). (h) Representative Western blot analysis to examine the expression level of BCR::ABL1-p190 protein in HEK 293T cells expressing tiled crRNAs targeting BCR::ABL1-p190 transcripts 24 h post-transfection; N = 3. (See uncropped blots in the Source file). (i) Schematic of BCR::ABL1 dependent phosphorylation of ERK and Stat proteins, and inhibition of BCR::ABL1 oncogenic activity with imatinib, a potent competitor of ATP binding to ABL kinase. (j) Representative Western blot analysis examining the suppression of BCR::ABL1 expression and the subsequent inhibition of STAT5 and ERK phosphorylation in HEK 293T cells expressing BCR::ABL1-p190, Psp Cas13b and either NT or crRNA targeting the BCR::ABL1 24 h post-transfection. HEK 293T cells expressing BCR::ABL1-p190 and Psp Cas13b treated with 1µM imatinib for 4 hours were used as a positive control. Parental cells are HEK 293T cells transfected with Psp Cas13b, NT and a random control plasmid. This condition shows the baseline expression of pSTAT5 and pERK in a BCR::ABL1 independent manner; N = 2. N is the number of independent biological experiments. Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Reporter Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Inhibition, Activity Assay, Binding Assay, Positive Control, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a–b) Comprehensive analysis of spacer-target interaction examining specificity and mismatch tolerance of Psp Cas13b crRNAs targeting the breakpoint region of BCR::ABL1-p190 transcript. The nucleotides in red highlight various mismatch positions in the spacer sequence. Data points in the graph are averages of mean fluorescence from 4 representative fields of views per condition imaged; N = 4. The data are represented in arbitrary units (A.U.). Errors are SEM and p -values of the one-way ANOVA test are indicated (95% confidence interval). (c) Western blot analysis examining the expression level of BCR::ABL1-p190 protein and phosphorylation status of STAT5 and ERK in HEK 293T cells expressing crRNAs with various mismatches 24 h post-transfection; N = 3. (See uncropped blots in the Source file). (d-f) Three colour fluorescence-based reporter assays to assess the specificity of crRNA targeting the breakpoint region of BCR::ABL1 in HEK 293T cells 48 h post-transfection. The schematics show (d) BCR::ABL1-mCherry mRNA, (e) ABL1-eGFP mRNA, and (f) BCR-TagBFP mRNA and their interaction with crBCR, crBCR::ABL1 and crABL1 crRNAs through full, partial, or no spacer-target basepairing. The quantifications of the silencing of (d) BCR::ABL1-mCherry, (e) ABL1-eGFP and (f) BCR-TagBFP. Data points are averages of normalised mean fluorescence from 4 representative fields of view per condition imaged. The data are represented in arbitrary units (A.U.). Errors are SEM and p -values of the one-way ANOVA test are indicated (95% confidence interval). N = 3. The right panels show representative western blot analyses to examine the expression levels of ΔBCR-ABL, ΔABL1, and ΔBCR proteins from data in d-f; N = 3 (See uncropped blots in the Source file).

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Sequencing, Fluorescence, Western Blot, Expressing, Transfection

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) Representative fluorescence microscopy images show the silencing efficiency of crBCR, crBCR::ABL1 and crABL1 crRNAs targeting BCR::ABL1-mCherry, ABL1-eGFP and BCR-TagBFP transcripts in HEK 293T cells. Scale bar = 100 μm, N = 3. Unprocessed representative images are provided in the Source Data file. (b) Assessment of potential Psp Cas13b (lel) and Rfx Cas13d (right) collateral activity through the analysis of ribosomal RNA (28S and 18S) degradation in the presence of target-free (NT) or target-bound (crBCR::ABL1) Cas13 nuclease, N =3. N is the number of independent biological experiments. (c) A volcano plot of transcripts showing the log 2 -fold change (FC) in HEK 293T cells expressing Psp Cas13b with a T crRNA versus an NT crRNA. Each data point represents a gene, and genes with log 2 FC > 1 (upregulation, indicated in red) or log 2 FC < -1 (downregulation, indicated in blue), and p-value < 0.05 are considered as significant differentially expressed transcript, N =3. (d) Counts per million (CPM) of split reads that mapped to BCR::ABL1 transcript in HEK 293T cells treated with Psp Cas13b or imatinib (lel). The frequency of split and contiguous reads that mapped to BCR::ABL1 transcript in parental and TKI-resistant K562 cells treated with Psp Cas13b or imatinib (right). (e) RT-qPCR assays to measure the Rfx Cas13d-mediated knockdown of BCR-ABL1-p190 and eGFP using primers shown in , N =3. Data are normalized means and errors are SEM. Results are analysed by unpaired two-tailed Student’s t-test (95% confidence interval). N represents the number of biological replicates). Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Fluorescence, Microscopy, Activity Assay, Expressing, Quantitative RT-PCR, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: RNA sequencing assays were performed in HEK 293T cells transfected with plasmids coding Psp Cas13b, BCR::ABL1-p190-IRES-eGFP and either a crRNA targeting the breakpoint of BCR::ABL1-p190 transcript (T) or non-targeting (NT) crRNA. RNA was extracted 48 hours post-transfection. (a) A linear regression plot showing log 2 -counts per million (CPM) of transcripts and (b) a mean-difference plot (MD-plot) showing the log 2 -fold change(FC) and average abundance of transcripts in HEK 293T cells expressing Psp Cas13b with a T crRNA versus an NT crRNA. Each data point represents a gene, and genes with log 2 FC > 1 (upregulation, indicated in red) or log 2 FC < -1 (downregulation, indicated in blue), and p-value < 0.05 are considered as significant differentially expressed transcripts. N =3; ( N represents the number of biological replicates). (c) Representative Arriba fusion plots showing the read alignments against the BCR::ABL1 transcript in HEK 293T cells expressing Psp Cas13b with either an NT (top) or a T (bosom) crRNA. The breakpoint region is highlighted with a dashline box. (d) Counts per million (CPM) (lel) and frequency (right) of split reads exclusively mapped to BCR::ABL1 breakpoint (filter criteria shown in the method section) in HEK 293T cells treated with Psp Cas13b or imatinib. (e) Schematic showing the design of RT-qPCR primers binding different regions on BCR::ABL1-p190-IRES-eGFP transcript to determine the fate of cleavage RNA fragments (top). RT-qPCR assays (bosom) to measure the expression levels of BCR::ABL1-p190 and eGFP using primers shown in the top schematic. N =3. Data are normalised means and errors are SEM. Results are analysed by unpaired two-tailed Student’s t-test (95% confidence interval). N represents the number of biological replicates). Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: RNA Sequencing Assay, Transfection, Expressing, Quantitative RT-PCR, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) The schematic illustrates the binding site of targeting (T) crRNA (crRNA0 in this case) to the breakpoint of BCR::ABL1-p210 mRNA, and experimental pipelines to assess Psp Cas13b - mediated silencing of BCR::ABL1 p210 in K562 cells using crRNA0. (b-c) Representative histograms of pSTAT5 and pERK staining in K562 cells expressing Psp Cas13/crRNA (b) or treated with imatinib (c) , N =3. (d) Sanger sequencing confirmed the heterozygous expression of T315I mutation of BCR::ABL1 in TKI-resistance K562 cells. (e-f) Representative fluorescence microscopy images (e) and histograms (f) show the expression of Psp Cas13b-mFlame in K562 cells expressing a non-targeting (NT) control crRNA versus a crRNA0 targeting (T) crRNA, N = 3. (g) Representative microscopy images show K562 cell density, parental (top) and TKI-resistant (bosom), at 96h time points post Psp Cas13b/crRNA delivery in cells expressing either NT or crRNA0 targeting (T) the BCR::ABL1-p210 mRNA. Scale bar = 300 μm. Similar results were obtained in N =3. Unprocessed representative images are provided in the Source Data file. N is the number of independent biological experiments. Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Binding Assay, Staining, Expressing, Sequencing, Mutagenesis, Fluorescence, Microscopy, Control

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) RT-PCR assays to measure the expression levels of BCR::ABL1 p210, BCR and ABL1 mRNAs in K562 cells expressing non-targeting (NT) crRNA or crRNA0 targeting (T) BCR::ABL1 p210 mRNA, N =2. Data are normalized means and errors are SEM. (b) Intracellular cell flow cytometry measuring STAT5 and ERK phosphorylation levels in K562 cells following imatinib treatment or BCR::ABL1-p210 silencing with Psp Cas13b/T crRNA; N =3. Data are normalized mean fluorescence intensity (MFI) and errors are SEM; Results are analysed by unpaired two-tailed Student’s t-test (95% confidence interval). (c) Representative western blot analysis to examine the expression levels of BCR::ABL1-p210, untranslocated ABL1, and STAT5/ERK phosphorylation in K562 cells expressing NT or T crRNA; N =2 (See uncropped in the Source file). (d-e) Alarm blue assays to assess K562 cell proliferation following (d) imatinib treatment or (e) BCR::ABL1-p210 mRNA silencing with Psp Cas13b throughout a 96h time window (timepoint 0 is 24h post-transfection); N =3. Data are normalized means and errors are SEM; Results are analysed by unpaired Student’s T-test (95% confidence interval). (f) The schematic illustrates the imatinib-sensitivity or imatinib-resistance of wildtype (WT) BCR::ABL1 and T315I variants, respectively. (g) Representative western blot analysis to examine the suppression of T315I BCR::ABL1-p190 with Psp Cas13b. BCR::ABL1 expression and STAT5/ERK phosphorylation were examined in HEK 293T cells expressing WT or T315I BCR::ABL1 variants, Psp Cas13b, and either NT or T crRNAs (crRNA-12, crRNA0 and crRNA+12 previously assessed in ) 24 h post-transfection. HEK 293T cells expressing BCR::ABL1 variants and Psp Cas13b were treated with 1µM imatinib for 4 hours as a positive control. HEK 293T cells were transfected with Psp Cas13b, NT and a control plasmid, which shows the baseline expression of pSTAT5 and pERK; N = 3 (See uncropped in the Source file). (h) Representative western blot analysis to confirm the imatinib-sensitivity or imatinib-resistance of parental and TKI-resistant K562 cells, respectively. N = 3 (See uncropped in the Source file). (i) Alamar blue assays to assess proliferation of TKI-resistant K562 cells following imatinib treatment throughout a 96h time window; N =3. Data are normalized means and errors are SEM; Results are analysed by unpaired Student’s T-test (95% confidence interval). (j) The schematic illustrates the experimental pipelines to assess Psp Cas13b - mediated silencing of BCR::ABL1-p210 in parental or TKI-resistant K562 cells using T crRNA (crRNA0). (k) Representative western blot analysis to examine the expression levels of Psp Cas13b BCR::ABL1-p210 and tyrosine phosphorylation in K562 cells expressing either NT or T crRNA 72 h post-transfection. K562 cells treated with imatinib or DMSO for 48h were used as controls; N =3 (See uncropped in the Source file). (l) Representative western blot analysis to examine the expression levels of BCR::ABL1-p210 and STAT5/ERK phosphorylation in K562 cells expressing either NT or T crRNA 48, 72 and 96 h post-transfection; N =3 (See uncropped in the Source file). (m) Alamar blue assays to assess proliferation of K562 cells following BCR::ABL1-p210 mRNA silencing with Psp Cas13 throughout a 96h time window (timepoint 0 is 24h post-transfection); N =3. Data are normalized means and errors are SEM; Results are analysed by unpaired Student’s T-test (95% confidence interval). (n) Representative FACS plots show K562 cells, parental (top) and TKI-resistant (bosom) expressing NT or T crRNA for 72 hours, then stained with Annexin V (Pacific Blue) and 7-ADD. Similar results were obtained in N =3. N is the number of independent biological replicates. Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Fluorescence, Two Tailed Test, Western Blot, Transfection, Positive Control, Control, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) Schematic of RNA sequencing and mass spectrometry assays to investigate the downstream effects of BCR::ABL1 silencing by Psp Cas13b in both parental and TKI-resistant K562 cells. The cells were nucleofected with Psp Cas13b-mFlame mRNA and either a targeting (T) or non-targeting (NT) crRNA plasmid. RNA and proteins were extracted 72 hours post-transfection. Cells treated with imatinib or DMSO for 48 hours were used as controls. (b, c) Volcano plots of RNA-sequencing analysis of the transcriptome of parental (top) and T315I (bosom) K562 cells expressing (b) Psp Cas13b or (c) treated with imatinib. Each data point represents a transcript, and transcripts with log 2 FC > 1 (upregulation, shown in red) or log 2 FC < -1 (downregulation, shown in blue), and a Benjamini-Hochberg adjusted p-value < 0.05 are considered significant differentially expressed transcripts. N =3. (d) Heatmap for 364 common transcripts differentially expressed in Psp Cas13b treated cell lines (parental or TKI-resistant) and imatinib-treated parental cells. (e) Gene ontology analysis for differentially expressed proteins shown in mass spectrometry data in and . (f) Gene ontology analysis for the 32 common differentially expressed proteins (25 upregulated and 7 downregulated) in Psp Cas13b/T crRNA expressing K562 (parental or TKI-resistant) cells and imatinib-treated parental cells shown in the Venn diagram . The gradient colour scheme represents the averaged log 2 FC of each protein’s expression in Psp Cas13b/T crRNA expressing K562 (parental or TKI-resistant) or imatinib-treated parental cells relative to their corresponding control. (g) Heatmap for 16 genes differentially expressed in cell lines (parental or TKI-resistant) treated with Psp Cas13b and parental cells treated with imatinib compared to their respective control groups. These genes were confidently detected by both RNA sequencing and mass spectrometry across all four comparison groups. (h) Gene ontology analysis for the 16 genes shown in the heatmap f . LCP2, SMIM1, and FN3K are not shown, as a minimum of two genes in each pathway is required. (i) STRING Protein-protein interaction networks functional enrichment analysis for the 16 genes shown in the heatmap g . N represents the number of biological replicates. Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: RNA Sequencing Assay, Mass Spectrometry, Plasmid Preparation, Transfection, Expressing, Control, Comparison, Functional Assay

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a-b) Representative Arriba fusion plots (right) and Sashimi plots showing the read alignments against the BCR-ABL1 transcript in parental (a) and TKI-resistant (b) K562 cells expressing Psp Cas13b with either a non-targeting (NT) or a targeting (T) crRNA, or treated with either imatinib or DMSO. The breakpoint region is highlighted with a dashline box and the approximate cleavage site is indicated with a red arrow. (c) Counts per million (CPM) of non-canonical split reads that exclusively mapped to BCR::ABL1 breakpoint (filter criteria shown in the method section) in parental and TKI-resistant K562 cells treated with Psp Cas13b or imatinib. N represents the number of biological replicates). Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Expressing

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) Venn diagrams showing the number of overlapping and unique transcripts differentially expressed (defined by a log 2 fold change >1 or <-1, with an adjusted p-value < 0.05) in RNA-sequencing data from the imatinib or Psp Cas13b treated K562 cells relative to the corresponding control K562 cells. (b) Gene ontology analysis of the common 400 transcripts differentially expressed in Psp Cas13b treated cell lines (parental or TKI-resistant) and imatinib-treated parental cells shown in the Venn diagrams a . (c) The list of top 15 upregulated and downregulated proteins from the RNA sequencing analysis in parental and TKI-resistant K562 cells treated with Psp Cas13b or imatinib. (d) Gene ontology analysis for the top 60 up-regulated and 60 downregulated transcripts shown in the heatmap .

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: RNA Sequencing Assay, Control

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) Western Blot validation of BCR::ABL1 expression in TKI-resistant and parental K562 cells expressing Psp Cas13b with either a non-targeting (NT) crRNA or a targeting (T) crRNA, using the same lysates as those used for mass spectrometry analyses, N = 5 (See uncropped blots in the Source file). (b, c) Volcano plots of mass spectrometry analysis of the proteome of parental (top) and T315I (bosom) K562 cells expressing (b) Psp Cas13b or (c) treated with imatinib. Each data point represents a protein, and proteins with log 2 FC > 0.5 (upregulation, shown in red) or log 2 FC < -0.5 (downregulation, shown blue), and p-value < 0.05 are considered significant differentially expressed proteins. Common up or down-regulated proteins of Psp Cas13b-treated cells and imatinib-treated TKI-resistant K562 cells are labelled in bold in the plot. N =5. (d) Venn diagrams showing the number of overlapping and unique proteins differentially expressed (calculated as log 2 fold change >1 or <-1, with an adjusted p-value <0.05) in mass spectrometry data from the imatinib or Psp Cas13b treated K562 cells relative to the corresponding control K562 cells. (e) Heatmap for 115 proteins differentially expressed at log 2 FC >0.5 or <−0.5 in at least one of the Psp Cas13b treated cell lines (parental or TKI-resistant). The colour scheme represents the log 2 FC of each protein’s expression in Imatinib or Psp Cas13b treated K562 cells relative to their corresponding control. (f) STRING Protein-protein interaction networks functional enrichment analysis for the 32 common up (25) and down-regulated (7) proteins shown in the Venn diagrams in d and the chord diagram in . N is the number of independent biological experiments. Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Western Blot, Expressing, Mass Spectrometry, Control, Functional Assay

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a-b) The list of top 15 upregulated (a) and downregulated proteins (b) from the proteomic analysis in parental and TKI-resistant K562 cells treated with Psp Cas13b or imatinib. (c-d) Gene ontology analyses of upregulated (c) and downregulated (d) proteins in parental and TKI-resistant K562 Cells treated with Psp Cas13b or imatinib. Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques:

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) Venn diagrams display the number of overlapping and unique genes confidently detected by both RNA sequencing and mass spectrometry in K562 cells treated with imatinib or Psp Cas13b, as well as in the corresponding control cells. (b) Relationship between RNA and protein log 2 fold change (FC) levels of genes measured by RNA sequencing and mass spectrometry in K562 cells treated with imatinib or Psp Cas13b, relative to their corresponding control cells. Pearson correlation analyses were performed. r (correlation coefficient) and p- value (95% confidence interval) are indicated in each graph. Results are analysed by unpaired two-tailed Student’s t-test. Error bars are SEM. Source data are provided as a Source data file

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: RNA Sequencing Assay, Mass Spectrometry, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a) Representative western blot analysis to examine the suppression of C464W and T315I-C464W BCR::ABL1-p190 variants with Psp Cas13b. BCR::ABL1 expression and STAT5 phosphorylation were examined in HEK 293T cells expressing WT, C464W or T315I-C464W BCR::ABL1 variants, Psp Cas13b, and either NT or T crRNAs (crRNA0 previously assessed in ) 24 h post-transfection. HEK 293T cells expressing BCR::ABL1 variants and Psp Cas13b were treated with 1µM asciminib for 4 hours as a positive control. Untransfected HEK 293T cells show the baseline expression of pSTAT5; N = 3 (See uncropped WB in the Source file). EGFP reporter assay to assess the silencing efficiency of tiled Psp Cas13b crRNAs incorporated with a 5’ GG motif with 2-nucleotide resolution targeting the breakpoint region of fusion transcripts EWSR1::FLI type 1 (EWSR1 Exon 7 to FLI1 Exon 6 translocation) (b) and type 2 (EWSR1 Exon 7 to FLI1 Exon 5 translocation) (c) . Data points in the graphs are averages of mean fluorescence from 4 representative fields of view per condition imaged. The data are represented in arbitrary units (A.U.). Errors are SEM.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Western Blot, Expressing, Transfection, Positive Control, Reporter Assay, Translocation Assay, Fluorescence

Journal: bioRxiv

Article Title: Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

doi: 10.1101/2025.02.10.637348

Figure Lengend Snippet: (a-c) Schematic (top) of predicted Psp Cas13b crRNAs targeting the breakpoint sequence of (a) RUNX1-RUNX1T1, (b) NPM-ALK and (c) SS18-SSX1/2 mRNAs, respectively. Quantifications (middle) of the silencing efficiency of predicted potent crRNAs targeting (a) RUNX1-RUNX1T1, (b) NPM-ALK and (c) SS18-SSX1/2 fusion transcripts in HEK 293T cells. Data points in the graph are averages of normalised mean fluorescence from 4 representative fields of view per experiment imaged in N = 3. The data are represented in arbitrary units (A.U.). Errors are SEM and p -values of the one-way ANOVA test are indicated (95% confidence interval). Representative western blot (bosom) analysis to examine the expression level of (a) RUNX1-RUNX1T1, (b) NPM-ALK and (c) SS18-SSX1/2 in HEK 293T cells; N =3 (See uncropped blots in the Source file). (d) Design principles for potent, specific, and personalised silencing of various gene fusion mRNAs with programmable Psp Cas13b nuclease. The lel panel shows the rational design of crRNAs targeting the breakpoint of gene fusion transcripts, which enables efficient and specific silencing of these oncogenic drivers, as well as drug-resistant fusion mutants. Psp Cas13b cleavage selectively trimmed a defined region near its binding site, leaving distal regions intact. The cleaved strands underwent RNA ligation, generating out-of-frame sequences that disrupted BCR::ABL1 translation. This targeting of the breakpoint strategy avoids the off-targeting of untranslocated gene partners that are expressed in normal tissues. The top right panel summarises spacer nucleotide positions that are important for the rational design of potent crRNA, which are integrated into our in-silico tool (hsps://cas13target.azurewebsites.net/) . The bosom right panel shows the versatility and therapeutic potential of Psp Cas13b to silence various ‘ undruggable ’ gene fusion transcripts and their oncogenic networks in a personalised manner through precise targeting of fusion’s breakpoint sequence that is exclusively expressed in tumour cells. N is the number of independent biological experiments. Source data are provided as a Source data file.

Article Snippet: For each well, a total of 100 ng DNA plasmids (22 ng of Psp Cas13b-NES-3xFLAG-T2A-BFP (addgene #173029) or pC0046-EF1a- Psp Cas13b-NES-HIV (addgene #103862), 22 ng crRNA plasmid, and 56 ng of the target gene plasmid) were mixed with 0.2 μL P3000 reagent in Opti-MEM Serum-free Medium (Thermo Fisher, 31985070) to a total of 5 μL (mix1).

Techniques: Sequencing, Fluorescence, Western Blot, Expressing, Binding Assay, Ligation, In Silico

Journal: Science Advances

Article Title: Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision

doi: 10.1126/sciadv.adl0731

Figure Lengend Snippet: ( A ) Silencing efficiency of four perfect-match, BRAF -targeting crRNAs at 48 hours after knock-in of WT (gray) or V600E (purple BRAF variants, normalized against crNT. ( B ) Systematic mutagenesis of nonselective cr BRAF -1 was used to engineer V600E -selective crRNAs. Sequences for each crRNA are shown below the bar plot; the “U” nucleotide (in red) indicates the uracil in the spacer sequence that base pairs with the target V600E but not with the WT BRAF RNA, and the colored nucleotides show the position of an additional mismatch introduced at various spacer locations through mutagenesis. Bar plot shows the silencing efficiency of cr BRAF -1 and its mutagenesis derivatives against BRAF - WT (gray) versus BRAF - V600E (purple), normalized against crNT at 48 hours after transfection. crE 5 -MM 8,15 and crE 5 -MM 11,15 (orange arrows) show the highest selectivity against BRAF V600E . ( C ) Delta silencing efficiencies between WT (gray) and V600E (purple) variants for the top-performing crRNAs, indicating the degree of SNV-specificity. Schematics depicting the sequence and base-pairing configuration of crE 5 -MM 8,15 and crE 5 -MM 11,15 with WT versus V600E -mutant BRAF mRNA targets are shown adjacent. ( D ) Dose-response curves derived from titration of parental cr BRAF -1, crE 5 -MM 8,15 , and crE 5 -MM 11,15 against WT (gray) or V600E (purple) BRAF at 48 hours after transfection. For all graphs in (A) to (C), individual data points show averaged fluorescence intensity from eight representative fields of view ( n = 3 independent experiments), and error bars show means ± SD. ( E ) Silencing efficiency assessed by Western blotting in HEK293T cells transfected with Psp Cas13b and full-length WT or SNV constructs. ( F ) Silencing efficiency assessed by RT-qPCR in cancer cell lines (A375 melanoma and HCT116 colorectal cancer), which endogenously express the SNV-containing oncogene (means ± SD from n = 3 independent experiments). Statistical significance was determined using unpaired t tests, where ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For 96-well plates, master mix A contained 25 ng of the Cas13 effector plasmid [ Psp Cas13b-NES-3xFLAG-T2A-BFP (Addgene #173029) or pLentiRNACRISPR-007- Rfx Cas13d (Addgene #138149)], target plasmid (50 ng of BRAF or 100 ng of KRAS expression plasmids), crRNA plasmid (0.1 to 100 ng), and 4% (v/v) P3000 enhancer reagent (Invitrogen) in Opti-MEM serum-free medium (Gibco), making up 5-μl total volume for 96-well plates.

Techniques: Knock-In, Mutagenesis, Sequencing, Transfection, Derivative Assay, Titration, Fluorescence, Western Blot, Construct, Quantitative RT-PCR

Journal: Science Advances

Article Title: Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision

doi: 10.1126/sciadv.adl0731

Figure Lengend Snippet: In this study, we show that comprehensive mutagenesis analysis of target-spacer interactions at single-nucleotide resolution reveals key spacer nucleotide positions intolerant to mismatches and the overall mismatch tolerance threshold of the target transcript. By designing crRNAs that follow these molecular principles, CRISPR-Cas13 can be reprogrammed for strong preferential silencing of SNV transcripts with single-base precision.

Article Snippet: For 96-well plates, master mix A contained 25 ng of the Cas13 effector plasmid [ Psp Cas13b-NES-3xFLAG-T2A-BFP (Addgene #173029) or pLentiRNACRISPR-007- Rfx Cas13d (Addgene #138149)], target plasmid (50 ng of BRAF or 100 ng of KRAS expression plasmids), crRNA plasmid (0.1 to 100 ng), and 4% (v/v) P3000 enhancer reagent (Invitrogen) in Opti-MEM serum-free medium (Gibco), making up 5-μl total volume for 96-well plates.

Techniques: Mutagenesis, CRISPR