Points of View on the Tools for Genome/Gene Editing

Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.

Sperm Oxidative Stress during In Vitro Manipulation and Its Effects on Sperm Function and Embryo Development

Reactive oxygen species (ROS) generated at low levels during mitochondrial respiration have key roles in several signaling pathways. Oxidative stress (OS) arises when the generation of ROS exceeds the cell’s antioxidant scavenging ability and leads to cell damage. Physiological ROS production in spermatozoa regulates essential functional characteristics such as motility, capacitation, acrosome reaction, hyperactivation, and sperm-oocyte fusion. OS can have detrimental effects on sperm function through lipid peroxidation, protein damage, and DNA strand breakage, which can eventually affect the fertility of an individual. Substantial evidence in the literature indicates that spermatozoa experiencing OS during in vitro manipulation procedures in human- and animal-assisted reproduction are increasingly associated with iatrogenic ROS production and eventual impairment of sperm function. Although a direct association between sperm OS and human assisted reproductive techniques (ART) outcomes after in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) is still a matter of debate, studies in animal models provide enough evidence on the adverse effects of sperm OS in vitro and defective fertilization and embryo development. This review summarized the literature on sperm OS in vitro, its effects on functional ability and embryo development, and the approaches that have been proposed to reduce iatrogenic sperm damage and altered embryonic development.

Suppressing the oxidation of LDL and DNA strand breakage of bioactives in dehulled and hull fraction of lentils

Hull/seed coat contains various plant cells that are responsible for sustaining the life of seeds in which phenolics perform significant roles in supporting their functions. In the present study, inhibitory activities against the oxidation of LDL and DNA of soluble- and insoluble-bound phenolics in the dehulled and hull fractions of lentils were determined. In addition, their α-glucosidase inhibitory activity was also assessed. The hulls possessed much higher levels of phenolics in both the soluble- and insoluble-bound forms than dehulled grain fractions, and this led to their potent activities in suppressing the oxidation of LDL and DNA and inhibiting α-glucosidase activity. The high bioactivities of hulls might be due to the existence of various types of cells such as palisade and parenchyma cells containing numerous phenolics for sustaining their functions. This study may help in a better understanding of the localization of soluble- and insoluble-bound phenolics in the structure of lentil.

APOBEC3B-dependent kataegis and TREX1-driven chromothripsis in telomere crisis

Chromothripsis and kataegis are frequently observed in cancer and can arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes1–5. Here we report on the mechanism underlying chromothripsis and kataegis using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes4, plays a prominent role in chromothriptic fragmentation. In absence of TREX1, the genome alterations induced by telomere crisis primarily involve Breakage-Fusion-Bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. In addition, APOBEC3B increased the frequency of chromothriptic fragmentation, possibly due to strand breakage after cytosine deamination. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.

Topoisomerase Inhibitors and Targeted Delivery in Cancer Therapy

DNA topoisomerases are enzymes that catalyze the alteration of DNA topology with transiently induced DNA strand breakage, essential for DNA replication. Topoisomerases are validated cancer chemotherapy targets. Anticancer agents targeting Topoisomerase I and II have been in clinical use and proven to be highly effective, though with significant side effects. There are tremendous efforts to develop new generation of topoisomerase inhibitors. Targeted delivery of topoisomerase inhibitors is another way to reduce the side effects. Conjugates of topoisomerases inhibitors with antibody, polymer, or small molecule are developed to target these inhibitors to tumor sites.

Local Strand-Breakage Detection in Multi-Strand Anchorage System Using an Impedance-Based Stress Monitoring Method—Feasibility Study

This study investigates the feasibility of impedance-based stress monitoring method for local-strand breakage detection in multi-strand anchorage systems. Firstly, stress fields of a multi-strand anchorage system are numerically analyzed to examine anchorage’s responses sensitive to local strand breakage. Secondly, an impedance-based stress monitoring technique via the PZT interface is outlined. Thirdly, a novel hoop-type PZT interface is designed for the multi-strands anchorage to monitor the stress variation induced by the strand breakage. Local dynamic responses of the hoop-type PZT interface are analyzed to predetermine the effective frequency ranges. Finally, the numerical feasibility of the proposed method is verified on a seven-strand anchorage system under various strand breakage cases. Variations in impedance responses are statistically quantified, and broken strands are localized by linear tomography analysis of damage indices. A lab-scale experiment is also conducted on a multi-strands anchorage to evaluate the realistic performance of the hoop PZT interface for impedance-based stress monitoring method.