genomic DNA

About: genomic DNA is a research topic. Over the lifetime, 15046 publications have been published within this topic receiving 663636 citations. The topic is also known as: genomic deoxyribonucleic acid & gDNA.

Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

Abstract: Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome.

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

Abstract: We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

Identification of a human achaete-scute homolog highly expressed in neuroendocrine tumors

Abstract: Basic helix-loop-helix transcription factors of the achaete-scute family are instrumental in Drosophila neurosensory development and are candidate regulators of development in the mammalian central nervous system and neural crest. We report the isolation and initial characterization of a human achaete-scute homolog that is highly expressed in two neuroendocrine cancers, medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). The human gene, which we have termed human achaete-scute homology 1 (hASH1), was cloned from a human MTC cDNA library. It encodes a predicted protein of 238 aa that is 95% homologous to mammalian achaete-scute homolog (MASH) 1, a rodent basic helix-loop-helix factor. The 57-residue basic helix-loop-helix domain is identical to that in the rodent gene, and the basic and helical regions, excluding the loop, are 72-80% identical to Drosophila achaete-scute family members. The proximal coding region of the hASH1 cDNA contains a striking 14-copy repeat of the triplet CAG that exhibits polymorphism in human genomic DNA. Thus, hASH1 is a candidate locus for disease-causing mutations via triplet repeat amplification. Analysis of rodent-human somatic cell hybrids permitted assignment of hASH1 to human chromosome 12. Northern blots revealed hASH1 transcripts in RNA from a human MTC cell line, two fresh MTC tumors, fetal brain, and three lines of human SCLC. In contrast, cultured lines of non-SCLC lung cancers and a panel of normal adult human tissues showed no detectable hASH1 transcripts. Expression of hASH1 may provide a useful marker for cancers with neuroendocrine features and may contribute to the differentiation and growth regulation of these cells.

Genomic targeting with purified Cre recombinase

Abstract: Purified Cre recombinase protein introduced directly into cultured mammalian cells by lipofection catalyzes both site-specific chromosomal integration of a co-transfected lox targeting vector and precise excision of genomic DNA flanked by directly repeated lox sites. This procedure eliminates the need to transfect cre expression plasmids to activate recombination at lox sites. We used this simplified procedure to investigate the effect on targeting efficiency of both lox vector design and chromosomal position of the lox target. We show that such chromosomal position effects can exert at least a 50-fold per lox target difference in targeting efficiency in a human osteosarcoma cell line.