肿瘤细胞治疗研究
英文原题：Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing.
CRISPR tiling筛选使得调控序列的特征描述成为可能，但受限于通过guide RNA测序和富集分析对编辑进行间接读取所带来的低分辨率。
CRISPR 平铺筛选已能够对调控序列进行表征，但受限于通过 guide RNA 测序和富集分析对编辑进行间接读取所导致的低分辨率。本研究引入了一种端到端实验检测方法和计算流程，其利用密集碱基编辑诱变后对内源性靶位点中 CRISPR 引入的等位基因进行靶向测序。作为概念验证，我们研究了一个推定的 CD19 增强子；CD19 是白血病中的免疫治疗靶点，并鉴定了对 CD19 调控至关重要的等位基因和单个核苷酸。我们的可视化工具揭示了与排名最高的核苷酸对应的转录因子基序。验证实验证实，MYB、PAX5 和 EBF1 结合位点的突变会降低 CD19 表达。关键的是，编辑 MYB 和 PAX5 基序赋予了对 CD19 CAR-T 细胞治疗的抗性，揭示了非编码变异如何驱动免疫治疗逃逸。总而言之，该方法在调控元件上实现了核苷酸分辨率的基因型-表型映射，超越了传统的基于 gRNA 的筛选。
CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.
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