spKAS-seq reveals R-loop dynamics using low-input materials by detecting single-stranded DNA with strand specificity

Wu, Tong; Lyu, Ruitu; He, Chuan

doi:10.1126/sciadv.abq2166

Cited by 16 publications

(13 citation statements)

References 58 publications

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“…This result is confirmed by overlaying the R-loop signals on single consecutive confocal z-sections and on the corresponding maximum intensity projection of control and TA-treated cells ( Supplementary Figure 1E ). The reduction in signal, as opposed to a complete loss, was also observed in previous sequencing-based studies ( 25 , 26 ), and is likely due to incomplete digestion of R-loops and to a subset of R-loops that are resistant to RNase H activity ( 27 ).…”

Section: Resultssupporting

confidence: 71%

Live-cell imaging unveils distinct R-loop populations with heterogeneous dynamics

Martin,

de Almeida,

Gameiro

et al. 2023

Nucleic Acids Research

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We have developed RHINO, a genetically encoded sensor that selectively binds RNA:DNA hybrids enabling live-cell imaging of cellular R-loops. RHINO comprises a tandem array of three copies of the RNA:DNA hybrid binding domain of human RNase H1 connected by optimized linker segments and fused to a fluorescent protein. This tool allows the measurement of R-loop abundance and dynamics in live cells with high specificity and sensitivity. Using RHINO, we provide a kinetic framework for R-loops at nucleoli, telomeres and protein-coding genes. Our findings demonstrate that R-loop dynamics vary significantly across these regions, potentially reflecting the distinct roles R-loops play in different chromosomal contexts. RHINO is a powerful tool for investigating the role of R-loops in cellular processes and their contribution to disease development and progression.

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Section: Resultssupporting

confidence: 71%

Live-cell imaging unveils distinct R-loop populations with heterogeneous dynamics

Martin,

de Almeida,

Gameiro

et al. 2023

Nucleic Acids Research

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“…The regions with excess ss-DNA hits were overlapped with predictions of Z-HUNT3 to define the regions of Z-DNA formation shown here in the KEx track. No filtering was performed for K-seq results with the strand-specific sequencing plus and minus tracks from HeLa cells revealing the R-loops formed when an RNA:DNA hybrid displaces an unpaired DNA stand from a double-stranded DNA helix ( Wu et al, 2022 ). The results indicate that the Z-DNA predictions for both Z-HUNT3 and Z-DNABERT align with only a small fraction of the ssDNA detected by K-seq.…”

Section: Resultsmentioning

confidence: 99%

“…We used existing data available from kethoxal-assisted sequencing studies ( Weng et al, 2020 ; Wu et al, 2020 ; Wu et al, 2022 ) and downloaded from GEO ( GSE202044 , GSE192822 , GSE139420 ). The Z-HUNT3 program is available at https://github.com/Ho-Lab-Colostate/zhunt .…”

Section: Methodsmentioning

confidence: 99%

Z-flipon variants reveal the many roles of Z-DNA and Z-RNA in health and disease

Umerenkov¹,

Herbert

Konovalov

et al. 2023

Life Sci. Alliance

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Identifying roles for Z-DNA remains challenging given their dynamic nature. Here, we perform genome-wide interrogation with the DNABERT transformer algorithm trained on experimentally identified Z-DNA forming sequences (Z-flipons). The algorithm yields large performance enhancements (F1 = 0.83) over existing approaches and implements computational mutagenesis to assess the effects of base substitution on Z-DNA formation. We show Z-flipons are enriched in promoters and telomeres, overlapping quantitative trait loci for RNA expression, RNA editing, splicing, and disease-associated variants. We cross-validate across a number of orthogonal databases and define BZ junction motifs. Surprisingly, many effects we delineate are likely mediated through Z-RNA formation. A shared Z-RNA motif is identified in SCARF2, SMAD1, and CACNA1 transcripts, whereas other motifs are present in noncoding RNAs. We provide evidence for a Z-RNA fold that promotes adaptive immunity through alternative splicing of KRAB domain zinc finger proteins. An analysis of OMIM and presumptive gnomAD loss-of-function datasets reveals an overlap of Z-flipons with disease-causing variants in 8.6% and 2.9% of Mendelian disease genes, respectively, greatly extending the range of phenotypes mapped to Z-flipons.

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“…The target DNA sequence is complementary to the first 20 or so nucleotides of the sgRNA, which are then followed by a sequence known as the PAM, which is complementary recognition site, directly adjacent to PAM, is also critical for subsequent Cas9 binding, R-loop formation, and initiation of nuclease activities of Cas9 (Wu et al 2022). These factors should be considered while designing a gRNA for targeting a specific sequence in the genome.…”

Section: Mechanism Of Off-target Effects In Crispr/cas9 Systemmentioning

confidence: 99%

Genome editing in cotton: challenges and opportunities

Khan

Ahmed

et al. 2023

J Cotton Res

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Cotton has enormous economic potential providing high-quality protein, oil, and fibre. A large increase in cotton output is necessary due to the world's changing climate and constantly expanding human population. In the past, conventional breeding techniques were used to introduce genes into superior cotton cultivars to increase production and to improve quality. The disadvantages of traditional breeding techniques are their time-consuming, reliance on genetic differences that are already present, and considerable backcrossing. To accomplish goals in a short amount of time, contemporary plant breeding techniques, in particular modern genome editing technologies (GETs), can be used. Numerous crop improvement initiatives have made use of GETs, such as zinc-finger nucleases, transcription-activator-like effector nucleases, clustered regularly interspaced palindromic repeats (CRISPR), and CRISPR-associated proteins systems (CRISPR/Cas)-based technologies. The CRISPR/Cas system has a lot of potential because it combines three qualities that other GETs lack: simplicity, competence, and adaptability. The CRISPR/Cas mechanism can be used to improve cotton tolerance to biotic and abiotic stresses, alter gene expression, and stack genes for critical features with little possibility of segregation. The transgene clean strategy improves CRISPR acceptability addressing regulatory issues associated with the genetically modified organisms (GMOs). The research opportunities for using the CRISPR/Cas system to address biotic and abiotic stresses, fibre quality, plant architecture and blooming, epigenetic changes, and gene stacking for commercially significant traits are highlighted in this article. Furthermore, challenges to use of CRISPR technology in cotton and its potential for the future are covered in detail.

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spKAS-seq reveals R-loop dynamics using low-input materials by detecting single-stranded DNA with strand specificity

Cited by 16 publications

References 58 publications

Live-cell imaging unveils distinct R-loop populations with heterogeneous dynamics

Live-cell imaging unveils distinct R-loop populations with heterogeneous dynamics

Z-flipon variants reveal the many roles of Z-DNA and Z-RNA in health and disease

Genome editing in cotton: challenges and opportunities

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