清脆的
移码突变
生物
Cas9
基因敲除
基因组编辑
计算生物学
遗传学
基因
分子生物学
DNA
外显子
作者
A. Smits,Frederik Ziebell,Gérard Joberty,Nico Zinn,William F. Mueller,Sandra Clauder‐Münster,Dirk Eberhard,Maria Fälth Savitski,Paola Grandi,Petra Jakob,Anne-Marie Michon,Hanice Sun,Karen Tessmer,Tilmann Bürckstümmer,Marcus Bantscheff,Lars M. Steinmetz,Gerard Drewes,Wolfgang Huber
出处
期刊:Nature Methods
[Springer Nature]
日期:2019-10-28
卷期号:16 (11): 1087-1093
被引量:158
标识
DOI:10.1038/s41592-019-0614-5
摘要
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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