Genome-wide mapping of native co-localized G4s and R-loops in living cells.

The interplay between G4s and R-loops are emerging in regulating DNA repair, replication, and transcription. A comprehensive picture of native co-localized G4s and R-loops in living cells is currently lacking. Here, we describe the development of HepG4-seq and an optimized HBD-seq methods, which robustly capture native G4s and R-loops, respectively, in living cells. We successfully employed these methods to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). We discovered that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes. We further demonstrated the helicase Dhx9 as a direct and major regulator that modulates the formation and resolution of co-localized G4s and R-loops. Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops -associated genes. Taken together, our work established that the endogenous co-localized G4s and R-loops are prevalently persisted in the regulatory regions of active genes and are involved in the transcriptional regulation of their linked genes, opening the door for exploring broader roles of co-localized G4s and R-loops in development and disease.

Expression, purification, and biochemical analysis of the RNA-DNA hybrid helicase Sen1/SETX from Chaetomium thermophilum.

Yeast Sen1 and its vertebrate ortholog Senataxin (also known as SETX) are RNA-DNA resolving helicases. Sen1 and SETX are implicated in multiple critical nuclear functions not limited to but including DNA replication and repair, RNA processing, and transcription. These> 200 kDa helicases have a two-domain architecture with an N-terminal regulatory helical repeat array linked to an SF1b helicase motor core via a variable sized central linker of low complexity sequence. Given the size of these proteins, production of milligram quantities of protein that is suitable for biochemical, biophysical, and protein structural analysis has been challenging. To overcome these limitations, we developed a robust selectable high-yield YFP-fusion protein expression method for Sen1 production in mammalian cells, followed by purification on a high-affinity YFP-binding camelid nanobody support. Herein, we detail methods and protocols for the expression and purification of recombinant Sen1 from the thermophilic fungus Chaetomium thermophilum, and the quantitative characterization of its RNA-DNA duplex resolution activity.

Simultaneous probing of transcription, G-quadruplex, and R-loop.

DNA and RNA can form various non-canonical secondary structures, including G-quadruplex (G4) and R-loops. These structures are considered transcriptional regulatory elements due to their enrichment at regulatory regions. During transcription, G-rich sequences in the non-template strand promote R-loop formation in the DNA template strand. These R-loops induce G4 structures in the non-template DNA strand, further stabilizing them. Additionally, the high rG: dC base-pairing within the R-loop contributes to the stability of DNA/RNA hybridization. Our previous study investigated the interplay between G4s and R-loops and its impact on transcription. We employed two techniques to demonstrate transcription-mediated G4 and R-loop formation. The single-molecule method allows us to detect intricate details of transcription initiation, elongation, and co-transcriptional R-loop and G4 formation. It provides a high-resolution view of the dynamic processes involved in transcriptional regulation. As an orthogonal approach, a gel-based assay enables the detection of the transcription-mediated R-loops and the RNA product. We can measure the progressive formation of R-loop and total RNA produced from transcription by analyzing gel electrophoresis patterns. In summary, these techniques provide valuable insights into the non-canonical nucleic acid structures and their impact on gene expression.

Transcription reprogramming and endogenous DNA damage.

Transcription reprogramming is essential to carry out a variety of cell dynamics such as differentiation and stress response. During reprogramming of transcription, a number of adverse effects occur and potentially compromise genomic stability. Formaldehyde as an obligatory byproduct is generated in the nucleus via oxidative protein demethylation at regulatory regions, leading to the formation of DNA crosslinking damage. Elevated levels of transcription activities can result in the accumulation of unscheduled R-loop. DNA strand breaks can form if processed 5-methylcytosines are exercised by DNA glycosylase during imprint reversal. When cellular differentiation involves a large number of genes undergoing transcription reprogramming, these endogenous DNA lesions and damage-prone structures may pose a significant threat to genome stability. In this review, we discuss how DNA damage is formed during cellular differentiation, cellular mechanisms for their removal, and diseases associated with transcription reprogramming.

RNA-DNA hybrids on protein coding genes are stabilized by loss of RNase H and are associated with DNA damages during S-phase in fission yeast.

RNA-DNA hybrid is a part of the R-loop which is an important non-standard nucleic acid structure. RNA-DNA hybrid/R-loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA-DNA hybrids and map their genomic locations in fission yeast cells. The RNA-DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild-type. The majority of RNA-DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S-phase and about 20% of the RNA-DNA hybrids overlap with Rad52 loci. During S-phase, more robust association of Rad52 with RNA-DNA hybrids was observed in the protein coding region than in M-phase. These results suggest that persistent RNA-DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S-phase, potentially through collision with DNA replication forks.

Inflammatory factor TNFα-induced circDMD mediates R-loop formation to promote tumorigenesis.

Chronic inflammation has been associated with the development of cancer in various anatomical sites. However, the crosstalk between inflammatory factors and circular RNAs (circRNAs) in tumorigenesis is unclear. Here, we revealed that circDMD was upregulated in Tumor necrosis factor alpha-like (TNFα)-induced HeLa cells. circDMD promoted the expression and nuclear translocation of Nuclear factor kappa B subunit (NF-κB) to activate downstream factors. circDMD absorbed miR-4711-5p to increase Lysine demethylase 5 A (KDM5A) expression, which reduced Suppressor of cytokine signaling 1 (SOCS1) to decrease the ubiquitination of Rela proto-oncogene (P65). In addition, circDMD promoted Fms related receptor tyrosine kinase 4 (VEGFR3) expression through the formation of an R-loop in its promoter. circDMD promoted tumor proliferation, metastasis and autophagy by activating the NF-κB pathways in vitro and in tumors derived from HeLa cells in vivo. Taken together, our results indicated that the expression of circDMD is induced by TNFα and contributes to tumorigenesis in cervical cancer (CC), which might help elucidate the regulatory effects of circRNAs on tumorigenesis.

Endonuclear Circ-calm4 regulates ferroptosis via a circR-Loop of the COMP gene in pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is a serious pulmonary vascular disease characterized by vascular remodeling. Circular RNAs (CircRNAs) play important roles in pulmonary hypertension, but the mechanism of PH is not fully understood, particularly the roles of circRNAs located in the nucleus. Circ-calmodulin 4 (circ-calm4) is expressed in both the cytoplasm and the nucleus of pulmonary arterial smooth muscle cells (PASMCs). This study aimed to investigate the role of endonuclear circ-calm4 in PH and elucidate its underlying signaling pathway in ferroptosis. Immunoblotting, quantitative real-time polymerase chain reaction (PCR), malondialdehyde (MDA) assay, immunofluorescence, iron assay, dot blot, and chromatin immunoprecipitation (ChIP) were performed to investigate the role of endonuclear circ-calm4 in PASMC ferroptosis. Increased endonuclear circ-calm4 facilitated ferroptosis in PASMCs under hypoxic conditions. We further identified the cartilage oligomeric matrix protein (COMP) as a downstream effector of circ-calm4 that contributed to the occurrence of hypoxia-induced ferroptosis in PASMCs. Importantly, we confirmed that endonuclear circ-calm4 formed circR-loops with the promoter region of the COMP gene and negatively regulated its expression. Inhibition of COMP restored the phenotypes related to ferroptosis under hypoxia stimulation combined with antisense oligonucleotide (ASO)-circ-calm4 treatment. We conclude that the circ-calm4/COMP axis contributed to hypoxia-induced ferroptosis in PASMCs and that circ-calm4 formed circR-loops with the COMP promoter in the nucleus and negatively regulated its expression. The circ-calm4/COMP axis may be useful for the design of therapeutic strategies for protecting cellular functionality against ferroptosis and pulmonary hypertension.

Pushing the TAD boundary: Decoding insulator codes of clustered CTCF sites in 3D genomes.

Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer-promoter (E-P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward- and outward-oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD-boundary-crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long-distance E-P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA-mediated TAD boundary function via R-loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E-P interactions is developmentally regulated.

Structural insights into the diversity and DNA cleavage mechanism of Fanzor.

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

Synergistic combination of RAD51-SCR7 improves CRISPR-Cas9 genome editing efficiency by preventing R-loop accumulation.

CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.

Experimentally altering microRNA levels in embryos alters adult phenotypes.

We previously identified a unique genetic feature of Autism Spectrum Disorder (ASD) in human patients and established mouse models, a low to very low level of six microRNAs, miR-19a-3p, miR-361-5p, miR-3613-3p, miR-150-5p, miR-126-3p and miR-499a-5p. We attempted to interfere experimentally in mice with two of them, miR19a-3p and miR499a-5p by microinjecting into zygote pronuclei either the complementary sequence or an excess of the microRNA. Both resulted in low levels in the tissues and sperm of the targeted microRNAs and their pri and pre precursors. This method stably modify predetermined levels of miRNAs and identify miRNA alterations that cause changes in autistic behavior and predispose the individual to an inherited disease. Excess miRNA results in single-stranded miRNA variations in both free and DNA-bound RNA (R-loop) fractions in mouse models thus appearing to affect their own transcription. Analysis of miRNAs fractions in human patients blood samples confirm low level of six microRNAs also in R-loop fractions.

The role of DEAD- and DExH-box RNA helicases in neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) represent a large group of disorders with an onset in the neonatal or early childhood period; NDDs include intellectual disability (ID), autism spectrum disorders (ASD), attention deficit hyperactivity disorders (ADHD), seizures, various motor disabilities and abnormal muscle tone. Among the many underlying Mendelian genetic causes for these conditions, genes coding for proteins involved in all aspects of the gene expression pathway, ranging from transcription, splicing, translation to the eventual RNA decay, feature rather prominently. Here we focus on two large families of RNA helicases (DEAD- and DExH-box helicases). Genetic variants in the coding genes for several helicases have recently been shown to be associated with NDD. We address genetic constraints for helicases, types of pathological variants which have been discovered and discuss the biological pathways in which the affected helicase proteins are involved.

Discovery of an 8-oxoguanine regulator PCBP1 inhibitor by virtual screening and its synergistic effects with ROS-modulating agents in pancreatic cancer.

Introduction: Drugs that target reactive oxygen species (ROS) metabolism have progressed the treatment of pancreatic cancer treatment, yet their efficacy remains poor because of the adaptation of cancer cells to high concentration of ROS. Cells cope with ROS by recognizing 8-oxoguanine residues and processing severely oxidized RNA, which make it feasible to improve the efficacy of ROS-modulating drugs in pancreatic cancer by targeting 8-oxoguanine regulators. Methods: Poly(rC)-binding protein 1 (PCBP1) was identified as a potential oncogene in pancreatic cancer through datasets of The Cancer Genome Atlas (TCGA) project and Gene Expression Omnibus (GEO). High-throughput virtual screening was used to screen out potential inhibitors for PCBP1. Computational molecular dynamics simulations was used to verify the stable interaction between the two compounds and PCBP1 and their structure-activity relationships. In vitro experiments were performed for functional validation of silychristin. Results: In this study, we identified PCBP1 as a potential oncogene in pancreatic cancer. By applying high-throughput virtual screening, we identified Compound 102 and Compound 934 (silychristin) as potential PCBP1 inhibitors. Computational molecular dynamics simulations and virtual alanine mutagenesis verified the structure-activity correlation between PCBP1 and the two identified compounds. These two compounds interfere with the PCBP1-RNA interaction and impair the ability of PCBP1 to process RNA, leading to intracellular R loop accumulation. Compound 934 synergized with ROS agent hydrogen peroxide to strongly improve induced cell death in pancreatic cancer cells. Discussion: Our results provide valuable insights into the development of drugs that target PCBP1 and identified promising synergistic agents for ROS-modulating drugs in pancreatic cancer.

Association of R-loop binding proteins with prognosis and anti-tumor drug sensitivity in lung adenocarcinoma: a bioinfor-matic study. / RçŽ¯ç»“åˆè›‹ç™½å¯¹è‚ºè ºç™Œæ‚£è€ é¢„åŽçš„é¢„æµ‹åŠè¯ç‰©æ•æ„Ÿæ€§åˆ†æž.

OBJECTIVES: To investigate the association of R-loop binding proteins with prognosis and chemotherapy efficacy in lung adenocarcinoma. METHODS: The data related to R-loop regulatory genes were obtained from literature of R-loop proteomics and relevant databases. We used 403 cases of lung adenocarcinoma in the Cancer Genome Atlas as training set, and two datasets GSE14814 and GSE31210 in Gene Expression Omnibus as validation sets. The weighted gene co-expression network analysis (WGCNA) was employed to identify R-loop genes with a significant impact on the clinical phenotype of lung adenocarcinoma. Least absolute shrinkage and selection operator (LASSO) regression analysis was utilized to eliminate genes exhibiting multicollinearity. A multivariate Cox regression analysis was employed to scrutinize clinical variables and R-loop characteristic genes that exert independent prognostic effects on patient survival. Subsequently, a risk score model was constructed. The predictive capacity of this model for the prognosis of patients was analyzed and validated. Additionally, the performance of risk model on the anti-tumor drug sensitivity was assessed. The mutations of R-loop genes were analyzed by maftools. The effect of PLEC expression on anti-tumor drug sensitivity was tested on non-small cell lung adenocarcinoma H1299 and A549 cells in vitro. RESULTS: A collection of 1551 R-loop genes were obtained, and 78 genes exhibited significant effects on the clinical phenotype shown on WGCNA. The LASSO regression analysis retained fourteen R-loop genes. A multivariate Cox regression analysis further identified three R-loop genes (HEXIM1, GLI2, PLEC) and a clinical variable (tumor grading) that were associated with patient prognosis. Risk prediction model was established according to the regression coefficients of each parameter. Kaplan-Meier survival analysis showed that the prognosis of high-risk group was significantly worse than that of low-risk group (P<0.01). The time-dependent ROC curve showed that the risk model had good predictive ability in both training and validation sets. Predictive analyses of anti-neoplastic drug sensitivity indicated a diminished responsiveness to both chemotherapy and targeted treatment drugs among high-risk patients. The expression of PLEC was strongly correlated with sensitivity to gefitinib, a classical EGFR inhibitor. CONCLUSIONS: R-loop binding proteins have been identified as significant determinants in the prognosis and therapeutic strategies for lung adenocarcinoma, which indicates that therapeutic interventions targeting these specific R-loop binding proteins might contribute to a better survival of the patients.

Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems.

The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.

Modulation of diverse biological processes by CPSF, the master regulator of mRNA 3' ends.

The cleavage and polyadenylation specificity factor (CPSF) complex plays a central role in the formation of mRNA 3' ends, being responsible for the recognition of the poly(A) signal sequence, the endonucleolytic cleavage step, and recruitment of poly(A) polymerase. CPSF has been extensively studied for over three decades, and its functions and those of its individual subunits are becoming increasingly well-defined, with much current research focusing on the impact of these proteins on the normal functioning or disease/stress states of cells. In this review, we provide an overview of the general functions of CPSF and its subunits, followed by a discussion of how they exert their functions in a surprisingly diverse variety of biological processes and cellular conditions. These include transcription termination, small RNA processing, and R-loop prevention/resolution, as well as more generally cancer, differentiation/development, and infection/immunity.

Senataxin mediates R-loop resolution on HPV episomes.

Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.

RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination.

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

Decoding high mobility group A2 protein expression regulation and implications in human cancers.

High Mobility Group A2 (HMGA2) oncofetal proteins are a distinct category of Transcription Factors (TFs) known as "architectural factors" due to their lack of direct transcriptional activity. Instead, they modulate the three-dimensional structure of chromatin by binding to AT-rich regions in the minor grooves of DNA through their AT-hooks. This binding allows HMGA2 to interact with other proteins and different regions of DNA, thereby regulating the expression of numerous genes involved in carcinogenesis. Consequently, multiple mechanisms exist to finely control HMGA2 protein expression at various transcriptional levels, ensuring precise concentration adjustments to maintain cellular homeostasis. During embryonic development, HMGA2 protein is highly expressed but becomes absent in adult tissues. However, recent studies have revealed its re-elevation in various cancer types. Extensive research has demonstrated the involvement of HMGA2 protein in carcinogenesis at multiple levels. It intervenes in crucial processes such as cell cycle regulation, apoptosis, angiogenesis, epithelial-to-mesenchymal transition, cancer cell stemness, and DNA damage repair mechanisms, ultimately promoting cancer cell survival. This comprehensive review provides insights into the HMGA2 protein, spanning from the genetic regulation to functional protein behavior. It highlights the significant mechanisms governing HMGA2 gene expression and elucidates the molecular roles of HMGA2 in the carcinogenesis process.

Genome Editing of Mammalian Cells Through RNA Transcript-Mediated Homologous Recombination Repair.

Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.