Biological plasticity rescues target activity in CRISPR knock outs

• Published in: Nature methods Vol. 16; no. 11; pp. 1087 - 1093
• Main Authors: Smits, Arne H., Ziebell, Frederik, Joberty, Gerard, Zinn, Nico, Mueller, William F., Clauder-Münster, Sandra, Eberhard, Dirk, Fälth Savitski, Maria, Grandi, Paola, Jakob, Petra, Michon, Anne-Marie, Sun, Hanice, Tessmer, Karen, Bürckstümmer, Tilmann, Bantscheff, Marcus, Steinmetz, Lars M., Drewes, Gerard, Huber, Wolfgang
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.11.2019; Nature Publishing Group
• Subjects: 631/1647/1511; 631/208/191/2018; 631/61/191; Amino acid sequence; Analysis; Bioinformatics; Biological activity; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Cell Cycle Proteins - genetics; CRISPR; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA (Cytosine-5-)-Methyltransferase 1 - genetics; DNA sequencing; DNMT1 protein; Exons; Frameshift mutation; Gene Knockout Techniques; Humans; Isoforms; Life Sciences; Mass spectrometry; Mass spectroscopy; Mutation; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - genetics; Phenotypes; Preservation; Protein expression; Proteins; Proteomics; Ribonucleic acid; RNA; RNA sequencing; Transcription Factors - genetics; Germany
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/s41592-019-0614-5

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. One third of verified gene knock outs with CRISPR still show residual protein expression owing to translation reinitiation or exon skipping. Several proteins are still functional. The authors call for a systematic analysis of protein levels after genome editing.