Fig. 7: Proteomic analysis reveals high specificity and a lack of collateral activity against endogenous proteins.
a, Diagramming (created with BioRender.com) MS assays for exploring the off-target effects and collateral activities of PspCas13b in HEK 293T cells with ectopic BCR-ABL1(p190)-IRES-eGFP mRNA as a target. The cells were transfected with PspCas13b, BCR-ABL1 and targeting (T) or NT crRNA plasmids. dPspCas13b and crRNA only (no PspCas13b) were used as controls. Proteins were extracted 48 h after transfection, followed by trypsin digestion and MS analysis. b,f,j, eGFP reporter assays to assess BCR-ABL1 silencing obtained with PspCas13b (b), dPspCas13b (f) or crRNA only (j). Data points are the averaged mean fluorescence from four representative fields of view per imaged condition (n = 3). Errors are the s.e.m. and P values from an unpaired two-tailed Student’s t-test are indicated (95% confidence interval). c,g,k, RT–qPCR assays measuring BCR-ABL1 knockdown obtained with PspCas13b (c), dPspCas13b (g) or crRNA only (k) (n = 3). Data are the normalized means and errors are the s.e.m. Results were analyzed by an unpaired two-tailed Student’s t-test and P values are indicated (95% confidence interval). d,h,l, Representative western blots examining the expression of PspCas13b and BCR-ABL1 proteins obtained with PspCas13b (d), dPspCas13b (h) or crRNA only (l). Right, the graphs quantify the BCR-ABL1 and PspCas13b protein levels, with each data point representing the ratio of BCR-ABL1 to β-actin or PspCas13b to β-actin normalized to the NT crRNA condition (h,l, n = 3; d, n = 5). Error bars represent the s.e.m. and P values were calculated by an unpaired two-tailed Student’s t-test (95% confidence interval). e,i,m, Left, volcano plots showing the proteome of cells expressing PspCas13b (e), dPspCas13b (i) or crRNA only (m). Data points represent proteins, with log2FC > 1 (upregulation, blue) or log2FC < −1 (downregulation, red) and P < 0.05 indicating significantly differential expression. BCR-ABL1 and eGFP are labeled. Right, linear regression plots from the same experiments. Results were analyzed by an unpaired two-tailed Student’s t-test (n = 5).
