Gene knock outs (KOs) are efficiently engineered through CRISPR–Cas9-induced frameshift mutations. While the efficiency of DNA editing 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 sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR–Cas9-generated KO lines is necessary for phenotype interpretation.
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The transcriptomics and proteomics data of the 19 KO lines shown in Fig. 1b and the NGLY1 KO lines were deposited to publicly available repositories. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE57 partner repository with the dataset identifier PXD010335. RNA-seq data were deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-7061. The data for the remaining 174 KO lines shown in Fig. 1a are available from the corresponding author upon request.
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We would like to thank D. Pavlinic, F. Jung and V. Benes for RNA-seq; J. Stuhlfauth and team for cell banking; M. Boesche and team for mass spectrometry analyses; S. Shimamura for cell culture and sample preparation; and the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Illumina Human Methylation arrays and related services. A.H.S. was supported by a fellowship from the EMBL Interdisciplinary Postdoc (EIPOD) Programme under a grant from the Marie Sklodowska-Curie Actions COFUND (664726). The NGLY1 work was supported by the Grace Science Foundation.
G.J., N.Z., D.E., M.F.S., P.G., M.B., G.D. are employees and/or shareholders of Cellzome and GlaxoSmithKline. T.B. was an employee of Horizon Genomics GmbH.
Peer review information Nicole Rusk was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–6 and Supplementary Tables 1 and 2
Residual RNA and protein levels for 193 KO cell lines.
Residual peptide levels aggregated per exon of four BRD4 KO replicates.
Residual peptide levels aggregated per exon for two DNMT1 KO lines.
Residual mRNA and protein levels, and deglycosylation quantification of two NGLY1-KO clones.
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Smits, A.H., Ziebell, F., Joberty, G. et al. Biological plasticity rescues target activity in CRISPR knock outs. Nat Methods 16, 1087–1093 (2019) doi:10.1038/s41592-019-0614-5