RNA測序和三級質譜相結合發現CRISPR失效原因

　　德國歐洲分子生物學實驗室Wolfgang Huber、Lars M. Steinmetz和葛蘭素史克公司Gerard Drewes等研究人員發現，生物可塑性能夠挽救CRISPR敲除中的靶標活性。2019年10月28日，《自然—方法學》在線發表了這項成果。

　　研究人員設計了一種實驗策略，將RNA測序和三級質譜相結合，以表征193個靶向136個不同基因的缺失（經過基因驗證），在HAP1細胞中這些缺失由CRISPR誘導的移碼產生。研究人員觀察到了大約三分之一靶標的殘余蛋白表達，水平從低到未變化不等；并確定了兩種因果機制，翻譯重新開始導致N末端被截斷的目標蛋白，或者跳過了被編輯的外顯子，產生內部序列缺失的蛋白亞型。對三個截短的靶標BRD4、DNMT1和NGLY1的詳細分析顯示，這些蛋白部分保留了功能。

　　這些結果表明，系統性表征CRISPR-Cas9產生的基因敲除細胞系中殘余蛋白表達或功能對于表型解釋是必要的。

　　據介紹,通過CRISPR–Cas9引起的移碼突變高效實現了基因敲除。盡管可以通過DNA測序方便地驗證DNA編輯的效率，但目前仍缺乏對蛋白質消除效率的系統理解。

　　附：英文原文

　　Title: Biological plasticity rescues target activity in CRISPR knock outs

　　Author: Arne H. Smits, Frederik Ziebell, Gerard Joberty, Nico Zinn, William F. Mueller, Sandra Clauder-Mnster, Dirk Eberhard, Maria Flth Savitski, Paola Grandi, Petra Jakob, Anne-Marie Michon, Hanice Sun, Karen Tessmer, Tilmann Brckstmmer, Marcus Bantscheff, Lars M. Steinmetz, Gerard Drewes, Wolfgang Huber

　　Issue&Volume: 2019-10-28

　　Abstract: 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.

　　DOI: 10.1038/s41592-019-0614-5

　　Source: https://www.nature.com/articles/s41592-019-0614-5

　　期刊信息

　　Nature Methods：《自然—方法學》，創刊于2004年。隸屬于施普林格·自然出版集團，最新IF：28.467

　　官方網址：https://www.nature.com/nmeth/

　　投稿鏈接：https://mts-nmeth.nature.com/cgi-bin/main.plex