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NGLY1 frameshift mutation in K562 cells results in a partially functional NGLY1 truncation. (a) Residual RNA and protein levels of two NGLY1 frameshift mutation clones. Residual RNA levels were determined from a single RNA-seq experiment, residual protein levels from all spectra assigned to the protein in a single mass spectrometry measurement. (b) Sashimi plots indicating exon usage and splicing of the NGLY1 transcript in these and the parental lines. (c) Deglycosylation-dependent Venus fluorescence assay results for the WT and NGLY1 frameshift mutation lines stably expressing the ddVenus reporter. The square panels show FACS plots, quantifications are shown in the lower right. zVAD-FMK: NGLY1 inhibitor. c15 and c20: clones with frameshift mutations in exons 1 and 3. m01: clone with a patient-derived 37 mutation. m03 and m14: clones with a mutation in exon 8.
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Gene knockouts (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While DNA editing efficiency is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here, we devised an experimental strategy combining RNA-seq and triple-stage mass spectrometry (SP...
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... extend our observations to a second cell type, we assessed frameshift KO mutations of the NGLY1 gene, which encodes a de-N -glycosylating enzyme, in K562 cells (Fig. 5). Two KO clones termed NGLY1 c15 and NGLY1 c20 , that resulted from expansion of a single cell, were isolated to enable comparison of different frameshift mutations. By MS3-based quantitative mass spectrometry, we did not detect residual target protein expression in the KO lines (Fig. 5a). Interestingly, RNA sequencing revealed skipping ...
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... which encodes a de-N -glycosylating enzyme, in K562 cells (Fig. 5). Two KO clones termed NGLY1 c15 and NGLY1 c20 , that resulted from expansion of a single cell, were isolated to enable comparison of different frameshift mutations. By MS3-based quantitative mass spectrometry, we did not detect residual target protein expression in the KO lines (Fig. 5a). Interestingly, RNA sequencing revealed skipping of exon 3, which in both clones contained the frameshift mutation (Fig. 5b). Skipping of this exon results in an in-frame deletion of 246 bp, leading to a truncated protein missing Glycine 74 to Threonine 155. Although not detected by mass spectrometry, the expression of the in-frame ...
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... from expansion of a single cell, were isolated to enable comparison of different frameshift mutations. By MS3-based quantitative mass spectrometry, we did not detect residual target protein expression in the KO lines (Fig. 5a). Interestingly, RNA sequencing revealed skipping of exon 3, which in both clones contained the frameshift mutation (Fig. 5b). Skipping of this exon results in an in-frame deletion of 246 bp, leading to a truncated protein missing Glycine 74 to Threonine 155. Although not detected by mass spectrometry, the expression of the in-frame exon-3-skipping transcript led us to investigate potential residual function of the target enzyme. To characterize the ...
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... transcript led us to investigate potential residual function of the target enzyme. To characterize the deglycosylation activity of NGLY1, we performed a deglycosylation-dependent Venus fluorescence assay followed by cell sorting 35 . Both NGLY1 KO lines showed deglycosylation activity, albeit reduced to 60% to 65% compared to WT cells (Fig. ...
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... the addition of the NGLY1 inhibitor zVAD-FMK 36 further reduced the Venus fluorescence in the two NGLY1 mutant lines (Fig. 5c). To rule out nonspecific effects of zVAD-FMK, we generated patient-derived 37 , termed m01, and frameshift, termed m03 and m14, mutations in NGLY1 exon 8, that completely abrogated deglycosylation activity (Fig. 5c). Indeed, zVAD-FMK treatment did not further reduce Venus fluorescence in the exon 8 edited clones (Fig. 5c). These ...
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... the addition of the NGLY1 inhibitor zVAD-FMK 36 further reduced the Venus fluorescence in the two NGLY1 mutant lines (Fig. 5c). To rule out nonspecific effects of zVAD-FMK, we generated patient-derived 37 , termed m01, and frameshift, termed m03 and m14, mutations in NGLY1 exon 8, that completely abrogated deglycosylation activity (Fig. 5c). Indeed, zVAD-FMK treatment did not further reduce Venus fluorescence in the exon 8 edited clones (Fig. 5c). These results indicate that the cells of the clones NGLY1 c15 and NGLY1 c20 had reduced but measurable NGLY1-based deglycosylation activity. ...
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... two NGLY1 mutant lines (Fig. 5c). To rule out nonspecific effects of zVAD-FMK, we generated patient-derived 37 , termed m01, and frameshift, termed m03 and m14, mutations in NGLY1 exon 8, that completely abrogated deglycosylation activity (Fig. 5c). Indeed, zVAD-FMK treatment did not further reduce Venus fluorescence in the exon 8 edited clones (Fig. 5c). These results indicate that the cells of the clones NGLY1 c15 and NGLY1 c20 had reduced but measurable NGLY1-based deglycosylation activity. ...
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