PO.MCB08.01 · 分子与细胞生物学
gRNA、表位和转录组的多组学单细胞组合索引揭示K562细胞中药物敏感性的调节因子
Multi-omic single-cell combinatorial indexing of gRNA, epitopes, and transcriptome uncovers modulators of drug sensitivity in K562 cells
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摘要 Abstract
中文摘要
鉴定调节药物反应的基因对于理解治疗耐药性和发现改善癌症治疗的新靶点至关重要。传统的混合CRISPR筛选基于对向导RNA（gRNA）丰度的批量测量，分辨率有限，且无法捕捉与基因扰动相关的转录组或蛋白质组变化。为克服这些局限性，我们开发了单细胞组合式向导RNA、表位和转录组跨整合细胞组学信号图谱测绘（scGET-MOSAIC）测序，这是一个基于Cas13的单细胞CRISPR筛选平台，可同时从单个细胞中捕获gRNA身份、转录组和表面蛋白表达。该方法将混合筛选的可扩展性与单细胞多组学的分子深度相整合，能够对大型可成药基因文库进行全面的功能询问。与传统的批量CRISPR筛选相比，scGET-MOSAIC不仅提供更广的gRNA覆盖度，还提供多模态表型谱，揭示每种扰动如何改变细胞状态。此外，该平台克服了其他单细胞CRISPR方法（如CROP-seq）中存在的基因文库大小限制，允许在单次实验中分析数量大幅增加的扰动。我们将scGET-MOSAIC测序应用于用酪氨酸激酶抑制剂伊马替尼（imatinib）处理的慢性髓性白血病细胞系K562。跨数千个受扰动细胞的单细胞分析揭示了与药物反应相关的独特转录和表面蛋白特征。通过整合分析，我们鉴定出基因X是一个增强伊马替尼敏感性的关键调节因子，提示其为克服耐药性的潜在联合靶点。总之，由Cas13介导的RNA靶向驱动的scGET-MOSAIC测序，建立了一个用于大规模、高内涵CRISPR筛选的通用框架，将遗传扰动与转录组和蛋白质组表型联系起来。通过直接扰动RNA，该平台能够探索编码和非编码基因的功能，扩展了功能基因组学发现癌症中新型治疗脆弱性的能力。
查看英文原文 English abstract
Identifying genes that modulate drug response is critical for understanding therapeutic resistance and discovering new targets to improve cancer therapy. Conventional pooled CRISPR screens, based on bulk measurements of guide RNA (gRNA) abundance, provide limited resolution and cannot capture transcriptomic or proteomic changes associated with gene perturbations. To overcome these limitations, we developed Single-cell combinatorial Guide RNA, Epitope, and Transcriptome Mapping Of Signals Across Integrated Cellular omics (scGET-MOSAIC) sequencing, a Cas13-based single-cell CRISPR screening platform that simultaneously captures gRNA identity, transcriptome, and surface protein expression from individual cells. This approach integrates the scalability of pooled screening with the molecular depth of single-cell multi-omics, enabling comprehensive functional interrogation of large druggable gene libraries. Compared to traditional bulk CRISPR screens, scGET-MOSAIC provides not only broader gRNA coverage but also multi-modal phenotypic profiles that reveal how each perturbation alters cellular states. Furthermore, the platform overcomes gene library size constraints found in other single-cell CRISPR methods, such as CROP-seq, allowing a substantially higher number of perturbations to be analyzed in a single experiment. We applied scGET-MOSAIC sequencing to the chronic myeloid leukemia cell line K562 treated with the tyrosine kinase inhibitor imatinib. Single-cell analysis across thousands of perturbed cells revealed distinct transcriptional and surface protein signatures associated with drug response. Through integrative analysis, we identified gene X as a critical modulator that enhances imatinib sensitivity, suggesting a potential combinatorial target for overcoming drug resistance. In summary, scGET-MOSAIC sequencing, powered by Cas13-mediated RNA targeting, establishes a versatile framework for large-scale, high-content CRISPR screening that connects genetic perturbations to transcriptomic and proteomic phenotypes. By directly perturbing RNA, this platform enables the exploration of both coding and non-coding gene functions, expanding the capacity of functional genomics to uncover novel therapeutic vulnerabilities in cancer.
利益披露 Disclosure
S. Kang, None..
S. Lee, None..
B. Hwang, None.