Fig. 1 Schematic diagram of R-loop targeted editing and high-throughput screening system A: Schematic diagram of targeted R-loop editing technology based on CRISPR/dCas9 and RNase H1 enzyme. B: Workflow schematic of high-throughput screening system for R-loops based on sgRNA lentiviral library construction

Fig. 2 Establishment and identification of R-loop targeted editing technology in mammalian cells A: Schematics of two linearized lentiviral vector constructs; B: verification of dCas9 gene expression; C: confirmation of dCas9 protein and dCas9-RNaseH1 protein expression; D: validation of R-loop editing efficiency in the dCas9-RNaseH1-1 monoclonal cell line, *** P﹤0.001；**** P﹤0.000 1

Fig. 3 Cumulative distribution curve of sgRNA relative abundance

Fig. 4 Screening R-loop sites associated with drug resistance in HeLa cells A: Scatter plots showing changes in gene expression of sgRNA library in dCas9-RNaseH1 SAM cell lines before and after treatment with cisplatin and paclitaxel. B: Venn diagrams illustrating the impact of different drug treatments on gene expression of the sgRNA library. C: GO and KEGG enrichment analysis plots of R-loop functional sites in cisplatin-resistant and cisplatin-sensitive groups. D: GO and KEGG enrichment analysis plots of R-loop functional sites in paclitaxel-resistant and paclitaxel-sensitive groups

Fig. 5 Validation of candidate genes A: Cellular state diagram of dCas9-RNaseH1 cells transfected with different sgRNAs after treatment with different anti-tumor drugs. B: Cell count statistics of dCas9-RNaseH1 cell lines transfected with different sgRNAs after treatment with different anti-tumor drugs. C: Validation of R-loop editing efficiency in dCas9-RNaseH1 cell lines transfected with different sgRNAs. D: Validation of transcriptional expression differences in dCas9-RNaseH1 cell lines transfected with different sgRNAs. *P< 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.000 1