RNA测序和三级质谱相结合发现CRISPR失效原因

　　德国欧洲分子生物学实验室Wolfgang Huber、Lars M. Steinmetz和葛兰素史克公司Gerard Drewes等研究人员发现，生物可塑性能够挽救CRISPR敲除中的靶标活性。2019年10月28日，《自然—方法学》在线发表了这项成果。

　　研究人员设计了一种实验策略，将RNA测序和三级质谱相结合，以表征193个靶向136个不同基因的缺失（经过基因验证），在HAP1细胞中这些缺失由CRISPR诱导的移码产生。研究人员观察到了大约三分之一靶标的残余蛋白表达，水平从低到未变化不等；并确定了两种因果机制，翻译重新开始导致N末端被截断的目标蛋白，或者跳过了被编辑的外显子，产生内部序列缺失的蛋白亚型。对三个截短的靶标BRD4、DNMT1和NGLY1的详细分析显示，这些蛋白部分保留了功能。

　　这些结果表明，系统性表征CRISPR-Cas9产生的基因敲除细胞系中残余蛋白表达或功能对于表型解释是必要的。

　　据介绍,通过CRISPR–Cas9引起的移码突变高效实现了基因敲除。尽管可以通过DNA测序方便地验证DNA编辑的效率，但目前仍缺乏对蛋白质消除效率的系统理解。

　　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—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：28.467

　　官方网址：https://www.nature.com/nmeth/

　　投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex