METTL3-based epitranscriptomic editing screening identifies functional m6A sites in cancers [CRISPR screen_H358]

In this study, we used a targeted CRISPR/CasRx-METTL3 screen to identify m6A peaks that are critical for prostate and lung cancer. Overall design: The CRISPR screen employed in our pooled sgRNA library consisted of ~12,045 sgRNAs targeting m6A peaks identified from prostate cancer and lung cancer patients, with 5 sgRNAs per gene on average. The sgRNAs were designed using the "cas13design". Stable dCasRx-METTL3-expressing 22Rv1 and H358 cell line was generated. dCasRx-METTL3-expressing cell lines was infected with the library at an MOI of ~ 0.3 and coverage of 400x. 24h post-infection, cells were selected with blasticidin for 5 days and part of cells were sujected to DNA extraction as day 0. Other cells were cultured in dish for ~16 days (day 16), maintaining 400x coverage prior to our screening assay. Genomic DNA was extracted from replicate samples and sgRNA inserts were amplified by PCR. The input amount of genomic DNA was calculated to achieve 400x coverage of the library and resulting libraries were sequenced on an Illumina HiSeq500.

本研究采用靶向CRISPR/CasRx-METTL3筛选策略，旨在鉴定对前列腺癌与肺癌发生发展至关重要的m6A峰（m6A peaks）。整体实验设计如下：本研究所使用的混合sgRNA（single guide RNA）文库CRISPR筛选体系，共包含约12045条靶向从前列腺癌及肺癌患者样本中鉴定得到的m6A峰的sgRNA，平均每个基因对应5条sgRNA。sgRNA的设计依托"cas13design"工具完成。我们成功构建了稳定表达dCasRx-METTL3的22Rv1与H358细胞系。将该sgRNA文库以感染复数（MOI）约0.3、测序覆盖度400×的条件感染上述细胞系。感染24小时后，采用杀稻瘟菌素（blasticidin）对细胞进行5天的筛选，收取部分细胞提取基因组DNA，记为第0天样本。剩余细胞继续在培养皿中培养约16天（第16天），全程维持400×的测序覆盖度，直至开展后续筛选实验。从重复样本中提取基因组DNA，通过聚合酶链式反应（PCR）扩增sgRNA插入片段。通过计算确定基因组DNA的投入量，以确保文库覆盖度达到400×，最终构建的测序文库在Illumina HiSeq500测序平台上完成测序。