Sequencing uracil in DNA at single-nucleotide resolution

As an aberrant base in DNA, uracil is generated by dUMP misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution or specificity. Here, we present a UNG-independent Single-Nucleotide resolution Uracil Sequencing (SNU-seq) method utilizing the UdgX protein which specifically excises the uracil and forms a covalent bond with the resulting deoxyribose. SNU-seq was validated on synthetic DNA and applied to mammalian genomes. We found that the uracil content of pemetrexed-treated cells fluctuated along with DNA replication timing. We also accurately detected uracil introduced through cytosine deamination by the cytosine base editor (nCas9-APOBEC) and verified uracil occurrence in "WRC" motif within Activation-Induced Cytidine Deaminase (AID) hotspot regions in CSR-activated UNG-/- B cells.

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CRISPR-BEST: a highly efficient DSB-free base editor for filamentous actinomycetes

10.1101/582403 ◽

2019 ◽

Cited By ~ 2

Author(s):

Yaojun Tong ◽

Helene L. Robertsen ◽

Kai Blin ◽

Andreas K. Klitgaard ◽

Tilmann Weber ◽

...

Keyword(s):

Genome Instability ◽

Cytidine Deaminase ◽

Double Strand Breaks ◽

Antimicrobial Compounds ◽

Dna Double Strand Breaks ◽

Single Nucleotide ◽

Strand Breaks ◽

Genetic Manipulations ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractFilamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.

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Faculty Opinions recommendation of RNA structure analysis at single nucleotide resolution by selective 2'-hydroxyl acylation and primer extension (SHAPE).

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1024946.293765 ◽

2005 ◽

Author(s):

Douglas Turner

Keyword(s):

Structure Analysis ◽

Rna Structure ◽

Primer Extension ◽

Single Nucleotide ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

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Faculty Opinions recommendation of Mapping Causal Variants with Single-Nucleotide Resolution Reveals Biochemical Drivers of Phenotypic Change.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732571454.793543140 ◽

2018 ◽

Author(s):

Michael Travisano

Keyword(s):

Phenotypic Change ◽

Single Nucleotide ◽

Causal Variants ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

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Faculty Opinions recommendation of Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.734563155.793554762 ◽

2019 ◽

Author(s):

Anthony Maxwell

Keyword(s):

Escherichia Coli ◽

Dna Gyrase ◽

Cleavage Sites ◽

Single Nucleotide ◽

Resolution Mapping ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

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Light–Nucleotide versus Ion–Nucleotide Interactions for Single-Nucleotide Resolution

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.0c10759 ◽

2021 ◽

Author(s):

Mohsen Farshad ◽

Jayendran C. Rasaiah

Keyword(s):

Single Nucleotide ◽

Nucleotide Interactions ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

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Chemical probing of the homopurine·homopyrimidine tract in supercoiled DNA at single-nucleotide resolution

FEBS Letters ◽

10.1016/0014-5793(88)80102-9 ◽

1988 ◽

Vol 234 (2) ◽

pp. 295-299 ◽

Cited By ~ 26

Author(s):

M. Vojtíšková ◽

S. Mirkin ◽

V. Lyamichev ◽

O. Voloshin ◽

M. Frank-Kamenetskii ◽

...

Keyword(s):

Single Nucleotide ◽

Supercoiled Dna ◽

Chemical Probing ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

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Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map

Nature Protocols ◽

10.1038/s41596-021-00553-x ◽

2021 ◽

Author(s):

Alli L. Gombolay ◽

Francesca Storici

Keyword(s):

Genomic Dna ◽

Single Nucleotide ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

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Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq

BMC Genomics ◽

10.1186/1471-2164-12-479 ◽

2011 ◽

Vol 12 (1) ◽

Cited By ~ 45

Author(s):

Yi Wang ◽

Xiangzhen Li ◽

Yuejian Mao ◽

Hans P Blaschek

Keyword(s):

Clostridium Beijerinckii ◽

Rna Seq ◽

Single Nucleotide ◽

Resolution Analysis ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

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Identifying DNA mismatches at single-nucleotide resolution by probing individual surface potentials of DNA-capped nanoparticles

Nanoscale ◽

10.1039/c7nr05250b ◽

2018 ◽

Vol 10 (2) ◽

pp. 538-547 ◽

Cited By ~ 4

Author(s):

Hyungbeen Lee ◽

Sang Won Lee ◽

Gyudo Lee ◽

Wonseok Lee ◽

Kihwan Nam ◽

...

Keyword(s):

Kelvin Probe Force Microscopy ◽

Powerful Method ◽

Kelvin Probe ◽

Single Nucleotide ◽

Force Microscopy ◽

Surface Potentials ◽

Dna Mismatches ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

Here, we demonstrate a powerful method to discriminate DNA mismatches at single-nucleotide resolution from 0 to 5 mismatches (χ0 to χ5) using Kelvin probe force microscopy (KPFM).

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