基于R-loop靶向编辑技术的R-loop功能位点高通量筛选系统

doi:10.13560/j.cnki.biotech.bull.1985.2024-0613

摘要/Abstract

摘要：

【目的】开发哺乳动物细胞R-loop靶向编辑技术，探究其在肿瘤细胞耐药性研究中的应用。【方法】构建无酶切活性的Cas9与具有R-loop水解活性的RNase H1融合蛋白dCas9-RNaseH1表达载体，用于实现对R-loop的靶向编辑。在HeLa细胞中稳定表达该融合蛋白，建立R-loop靶向编辑细胞模型。转染覆盖全基因组转录起始区域的sgRNA文库，构建R-loop筛选细胞库，筛选影响紫杉醇和顺铂耐药性的R-loop功能位点。【结果】筛选获得744个影响HeLa细胞耐药性的R-loop功能位点，覆盖了细胞周期、凋亡、信号传导等关键生物通路。其中，26个位点使HeLa细胞对这两种药物产生耐药性，8个位点使其对这两种药物敏感，提示可能存在共享的生物通路。功能验证显示，部分R-loop功能位点通过调节相关基因（如ZBTB20、SPON2、ACTRT1等）的表达来影响HeLa细胞对抗肿瘤药物的敏感性。【结论】成功开发出适用于哺乳动物细胞的R-loop靶向编辑系统，并建立高通量筛选平台。

关键词: 高通量筛选, 耐药性, R-loop, 定点调控, CRISPR/dCas9

Abstract:

【Objective】 To develope R-loop targeted editing technology for mammalian cells and explore its applications in drug resistance of tumor cells. 【Method】 An expression vector was constructed to express a dCas9-RNaseH1 chimaera that combines catalytically dead Cas9 lacking endonuclease activity with RNase H1 possessing R-loop hydrolysis activity for R-loop targeted editing. This dCas9-RNaseH1 chimaera was transfected to HeLa cells and stably expressed to construct a model cell line. To create a cell library for R-loop screening, a genome-wide gRNA library covering transcription start sites was transfected to the model cell line, through which R-loop functional sites affecting resistance to paclitaxel and cisplatin were identified. 【Result】 Total 744 R-loop functional sites affecting HeLa cell drug resistance were identified, which cover key biological pathways such as cell cycle, apoptosis, and signal transduction. Among them, 26 sites confered resistance to two drugs, while 8 sites rendered sensitivity to both drugs, suggesting potential shared biological pathways. Functional validation revealed that certain R-loop sites modulated the expressions of relevant genes（e.g., ZBTB20, SPON2, ACTRT1）, significantly impacting HeLa cell sensitivity to anticancer drugs. 【Conclusion】 A R-loop targeted editing system is successfully developed and a high-throughput screening platform is established for mammalian cells.

Key words: high-throughput screening, drug resistance, R-loop, targeted regulation, CRISPR/dCas9

皮一飞, 宋新辉, 王淅琳, 李谨谨, 孙长斌, 徐炜. 基于R-loop靶向编辑技术的R-loop功能位点高通量筛选系统[J]. 生物技术通报, 2024, 40(10): 181-190.

PI Yi-fei, SONG Xin-hui, WANG Xi-lin, LI Jin-jin, SUN Chang-bin, XU Wei. High-throughput Screening System for Functional R-loop Loci Based on R-loop Targeted Editing Technology[J]. Biotechnology Bulletin, 2024, 40(10): 181-190.

图/表 8

图1 R-loop靶向编辑及高通量筛选系统示意图 A：基于CRISPR/dCas9与RNase H1酶的靶向编辑R-loop技术示意图；B：基于sgRNA慢病毒文库构建的R-loop高通量筛选系统流程示意图

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

表1 R-loop靶向调控技术构建所用引物序列

Table 1 Primer sequences used for constructing R-loop targeting regulatory technology

用途 Purpose	名称 Name	序列 Sequence（5'-3'）
构建慢病毒载体 Constructing lentiviral vector	BamH1-T2A-XbaI	CGGGATCCGAGGGAAGAGGTTCCCTGCTCACATGCGG AGACGTAGAAGAGAATCCCGGACCCTCTAGAGC
	RNaseH1-T2A-F	CGGGATCCTTCTATGCCGTGAGGAGGGGC
	RNaseH1-T2A-R	CGGGATCCTTCTATGCCGTGAGGAGGGGCGCTCTAGAGGTCATGGGTCCGGGATTCTCTTCTACGTCTCCGCATGTGAGCAGGGAACCTCTTCCCTCCCCTGAACCTGTACAGTTAATGTCTTCCGATTGTTTAGCTCCTTCTCTGGCT
dCas9 基因验证 Validating the dCas9 gene	dCas9-F	GAAGCAGCTTCGAGAAGAATCCCATCGAC
dCas9 基因验证 Validating the dCas9 gene	dCas9-R	GTCGATGGTGGTGTCAAAGTACTTGAAGGCG
验证编辑效率 Editing efficiency validation	RPL13A-1-sgR	GCGTGCGCAGAGATTCCCCA
	RPL13A-2-sgR	CGCGTGCGCAGAGATTCCCC
	RPL13A-3-sgR	GCTTCGACCAATGAAAACAC
	CALM3-1-sgR	CCGTGGGAGCCGCAGTGCGG
	CALM3-1-sgR	GATCCGTGGGAGCCGCAGTG
	CALM3-1-sgR	CGGGGCGCGGAGGGATCCG
	TFPT-1-sgR	GAGGAGAGGTGAGTGTGATG
	TFPT-2-sgR	GAGAGGAGAGGTGAGTGTGA
	TFPT-3-sgR	GCGACTAACGCTAGAAACAG
编辑效率验证DRIP-qPCR DRIP-qPCR for editing efficiency validation	RPL13A-F	AGGTGCCTTGCTCACAGAGT
	RPL13A-R	GGTTGCATTGCCCTCATTAC
	CALM3-F	GAGGAATTGTGGCGTTGACT
	CALM3-R	AGAGTGGCCAAATGAGCAGT
	TFPT-F	TCTGGGAGTCCAAGCAGACT
	TFPT-R	AAGGAGCCACTGAAGGGTTT
	RPL13A-F	AGGTGCCTTGCTCACAGAGT
	RPL13A-R	GGTTGCATTGCCCTCATTAC
	EGR1neg-F	GAACGTTCAGCCTCGTTCTC
	EGR1neg-R	GGAAGGTGGAAGGAAACACA
	SNRPNneg-F	GCCAAATGAGTGAGGATGG
	SNRPNneg-R	TCCTCTCTGCCTGACTCCAT

表2 sgRNA文库构建引物

Table 2 sgRNA library construction primer sequences

用途 Purpose	名称 Name	序列 Sequence（5'-3'）
sgRNA文库PCR sgRNA library PCR	Lib-plasmid-F	TCGTCGGCAGCGTCTGTGGAAAGGACGAAACACC
sgRNA文库PCR sgRNA library PCR	Lib-plasmid-R	GTCTCGTGGGCTCGGTGGCCAAGTTGATAACGGACTA
建库	N5XX	AATGATACGGCGACCACCGAGATCTACAC[IIIIIIII]TCGTCGGCAGCGTC
Library preparation	N7XX	CAAGCAGAAGACGGCATACGAGAT[IIIIIIII]GTCTCGTGGGCTCGG

表3 功能位点验证所用引物

Table 3 Primer sequences used for validation of functional sites

用途 Purpose	名称 Name	序列 Sequence（5'-3'）
合成R-loop功能位点sgRNA序列 Synthesize sgRNA sequences targeting R-loop functional sites	ZBTB20	AGGAGCAAAATGAAGCAGAA
	TUBAL3	TGGTCTCAGAGTAGCTGTGT
	TMEM176A	CCCAGTGACCCTCGCGCAGC
	TAF6	AGGGAGGTTCTGGTGGAGCT
	SPON2	CTGCGTGGGGTGGTGATGAA
	SOX14	TCTGCTGCTGATTGGCGCCC
	ACTRT1	ATTGGCTCAGAGGCCCCACC
	CARHSP1	GAACGCAGAGCGCGGGACGT
	CAMKK2	AGCCGGCGGGGGCGCGCAGG
	C4orf33	GAATCACCCTTCCTTCTTCT
	ZYG11A	AACAGAGAGGACCGTTAGAG
	ZFY	AGTTCGGAGCTGACAAAAAG
	CCDC71	GTTCTTCAGACTTCTGCACA
	CCDC30	TTTGAGACTTTGCTGTTGTT
	ZNF84	GTGCAGTCCCCGACTCGTCC
	TPP2	GAGGAGTCTGAGGACATTAC
	TXNRD1	TAGCCCGTGTATCTTCTTCA
	C9orf169	CTCAGGTGATCCGCCTGCCT
	CCM2	GGGAAGTAGGGAAAATGAGC
	C2orf74	CTAATTTCTCTCCATAACTG
	CCL22	GTGGAGAAATTCTCTTTGGC
	CA10	TGAGGCTGAGCGCGGCCATG
	CASP7	GTGCGTATGCTGTGGGGTTG
	CASC5	ATTGTGGGGGAGGTCTCCAC
qPCR验证 qPCR validation	ZBTB20-F-	GCGTGGACAGGATCTACTCG
	ZBTB20-R-	ATCGTCCTCCATCTCCTGCT
	SPON2-F	GCCAGAGCCCTGGCCAAATACAGCATC
	SPON2-R	TCCTCCACATGCTGTAGTCGGAGCTATG
	TPP2-F	TTCACACCCAGGCTCAAGAC
	TPP2-R	TGCAAATTTAAGGCCTCTCCC
	ZNF84-F	ATCTCCTACCCACGGCTCTT
	ZNF84-R	AAGGAGAAGAGCCGTGGGTA
	C4orf33-F	GTTGAACTTTGTCCCCACGGACAGC
	C4orf33-R	GAGATAAGCTTTGCCTTCCCATTTTGTCTCTC
	ACTRT1-F	TGTCATCAGCTCCGTCTTGG
	ACTRT1-R	TACCAGTCCACGCTCAATGG
	ZYF-F	TGTTCCTGATGACCCAGACTC
	ZYF-R	ATCTGGGACTGTGCAGTGTG
	C4orf74-F	GTTGAACTTTGTCCCCACGGACAGC
	C4orf74-R	GAGATAAGCTTTGCCTTCCCATTTTGTCTCTC
	CCL22-F	ACAGACTGCACTCCTGGTTG
	CCL22-R	GACGTAATCACGGCAGCAGA

图2 哺乳动物细胞 R-loop 靶向编辑技术的建立与鉴定 A：两种线性化慢病毒载体图；B：dCas9基因表达的鉴定；C： dCas9蛋白和dCas9-RNaseH1蛋白表达的鉴定；D：dCas9-RNaseH1-1 单克隆细胞系 R-loop 编辑效率验证

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

图3 sgRNA相对丰度累积分布曲线

Fig. 3 Cumulative distribution curve of sgRNA relative abundance

图4 筛选与 HeLa 细胞耐药性相关的R-loop位点 A：分别为顺铂和紫杉醇药物前后的dCas9-RNaseH1 SAM文库细胞系sgRNA文库基因表达变化散点图；B：不同药物处理对sgRNA文库基因表达影响的韦恩图；C：分别为顺铂药物处理耐药组和敏感组R-loop功能位点的GO和KEGG功能富集分析图；D：分别为紫杉醇药物处理耐药组和敏感组R-loop功能位点的GO和KEGG功能富集分析图

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

图5 候选基因的验证 A：不同抗肿瘤药物处理后不同sgRNA转染dCas9-RNaseH1细胞状态图；B：不同抗肿瘤药物处理后不同sgRNA转染dCas9-RNaseH1细胞系细胞数统计；C：不同sgRNA转染dCas9-RNaseH1细胞系R-loop编辑效率验证；D：不同sgRNA转染dCas9-RNaseH1细胞系转录表达差异验证

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

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