Dataset Information

Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

ABSTRACT: Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (γPNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the γPNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA-Guided T7EI (PG-T7EI) technology demonstrates that T7EI can be used as a programmable nuclease capable of generating single or multiple specific DSBs in vitro under a broad range of conditions and could be potentially applied for large-scale genomic manipulation. With no protospacer adjacent motif (PAM) constraints and featuring a compact protein size, our PG-T7EI system will facilitate and expand DNA manipulations both in vitro and in vivo, including cloning, large-fragment DNA assembly, and gene editing, with exciting applications in biotechnology, medicine, agriculture, and synthetic biology.

SUBMITTER: Aman R

PROVIDER: S-EPMC11014363 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

Aman Rashid R Syed Muntjeeb M MM Saleh Ahmed A Melliti Firdaws F Gundra Sivakrishna Rao SR Wang Qiaochu Q Marsic Tin T Mahas Ahmed A Mahfouz Magdy M MM

Nucleic acids research 20240401 6

Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (γPNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the γPNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA-Guided T7EI (PG-T7EI) technology demonstrate ...[more]

PMID: 38421613

Similar Datasets

Project description:ABSTRACT Aims: Double-stranded breaks (DSBs) cause genomic instability, a hallmark of aging, and activate the DNA damage response (DDR) and the cytosolic DNA-sensing proteins (CDSP) pathways. The activated pathways induce cell cycle arrest, inflammation, senescence, cell death, fibrosis, and organ dysfunction. The purpose of the study was to identify and characterize genome-wide DSBs at the nucleotide resolution in adult cardiac myocytes. Methods and Results: We identified the genome-wide DSBs in the genomes of ~ 6 million adult cardiac myocytes (1 million per sample) in the 3 wild-type and 3 myocyte-specific LMNA-deficient mice (Myh6-Cre:LmnaF/F) mice by END-Sequencing. We identified 6,774 differential DSBs between the wild-type and Myh6-Cre:LmnaF/F cardiac myocytes, which, except for 8, were exclusive to the Myh6-Cre:LmnaF/F genotype. The differential DSBs were enriched in the gene regions, transcription initiation sites, cardiac transcription factor motifs, and at the G quadruplex forming structures, suggesting the involvement of the transcriptional stress. Because LMNA regulates transcription, we performed a CUT&RUN assay with an anti-LMNA antibody (N=5) to test the role of transcriptional stress in an increased prevalence of DSBs. We identified an average of 818 genome-wide lamin-associated domains (LADs), which encompassed 30% of the mouse genome. LADs were associated with a 16-fold reduction in the transcript levels of protein-coding genes located at the LAD regions (N=3,975) as opposed to genes located at the non-LAD regions (N=~ 17,778). Consistent with increased transcription at the non-LAD regions DSBs were ~ 6-fold more prevalent in the non-LAD than LAD regions. Likewise, the loss of LAD was associated with a 2.3-fold higher prevalence of differential DSBs in the Myh6-Cre:LmnaF/F cardiac myocyte genomes, which were predominantly localized to the transcription start sites. Conclusions: To our knowledge, this is the first identification of the DSBs, a hallmark of aging, at the nucleotide resolution in the cardiovascular system. The findings implicate transcriptional stress in the pathogenesis of DSB and the protective role of LMNA against DSBs likely through transcriptional suppression. Given the ubiquitous nature of transcriptional stress, DSBs are expected to be pervasive and pathogenic through activation of the DDR and CDSP pathways not only in cardiovascular diseases but also in other conditions, including aging.

Dataset Information

Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

Publications

Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets