Gene Editing Institute, ChristianaCare, Newark, DE, USA.
Department of Medical and Molecular Sciences, University of Delaware, Newark, DE, USA.
Gene Ther. 2022 Jun;29(6):357-367. doi: 10.1038/s41434-022-00324-7. Epub 2022 Mar 22.
We have been developing CRISPR-directed gene editing as an augmentative therapy for the treatment of non-small cell lung carcinoma (NSCLC) by genetic disruption of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). NRF2 promotes tumor cell survival in response to therapeutic intervention and thus its disablement should restore or enhance effective drug action. Here, we report how NRF2 disruption leads to collateral damage in the form of CRISPR-mediated exon skipping. Heterogeneous populations of transcripts and truncated proteins produce a variable response to chemotherapy, dependent on which functional domain is missing. We identify and characterize predicted and unpredicted transcript populations and discover that several types of transcripts arise through exon skipping; wherein one or two NRF2 exons are missing. In one specific case, the presence or absence of a single nucleotide determines whether an exon is skipped or not by reorganizing Exonic Splicing Enhancers (ESEs). We isolate and characterize the diversity of clones induced by CRISPR activity in a NSCLC tumor cell population, a critical and often overlooked genetic byproduct of this exciting technology. Finally, gRNAs must be designed with care to avoid altering gene expression patterns that can account for variable responses to solid tumor therapy.
我们一直在开发 CRISPR 指导的基因编辑作为一种增强疗法，通过核因子红细胞 2 相关因子 2（NRF2）的遗传破坏来治疗非小细胞肺癌（NSCLC）。NRF2 促进肿瘤细胞对治疗干预的存活，因此其失活应该恢复或增强有效药物作用。在这里，我们报告 NRF2 破坏如何导致 CRISPR 介导的外显子跳跃形式的附带损伤。转录本和截断蛋白的异质群体对化疗产生不同的反应，这取决于缺失的功能域。我们鉴定和表征了预测和未预测的转录本群体，并发现几种类型的转录本通过外显子跳跃产生；其中一个或两个 NRF2 外显子缺失。在一个特定情况下，一个核苷酸的存在与否通过重组外显子剪接增强子（ESEs）决定一个外显子是否被跳过。我们从 NSCLC 肿瘤细胞群体中分离和表征 CRISPR 活性诱导的克隆多样性，这是这项令人兴奋的技术的一个关键且经常被忽视的遗传副产品。最后，gRNA 的设计必须小心，以避免改变基因表达模式，这些模式可能导致对实体瘤治疗的反应不同。
