Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common neurodegenerative diseases, and they affect millions of people worldwide, particularly older individuals. Therefore, there is a clear need to develop novel drug targets for the treatment of age-related neurodegenerative diseases. Emerging evidence suggests that mitochondrial dysfunction and reactive oxygen species (ROS) generation play central roles in the onset and progression of neurodegenerative diseases. Mitochondria are key regulators of respiratory function, cellular energy adenosine triphosphate production, and the maintenance of cellular redox homeostasis, which are essential for cell survival. Mitochondrial morphology and function are tightly regulated by maintaining a balance among mitochondrial fission, fusion, biogenesis, and mitophagy. In this review, we provide an overview of the main functions of mitochondria, with a focus on recent progress highlighting the critical role of ROS−induced oxidative stress, dysregulated mitochondrial dynamics, mitochondrial apoptosis, mitochondria-associated inflammation, and impaired mitochondrial function in the pathogenesis of age-related neurodegenerative diseases, such as AD and PD. We also discuss the potential of mitochondrial fusion and biogenesis enhancers, mitochondrial fission inhibitors, and mitochondria-targeted antioxidants as novel drugs for the treatment of these diseases.

The CRISPR-Cas system has transformed our ability to edit and modify genomes in eukaryotic cells, offering unmatched precision and broad applicability. By utilizing a programmable RNA protein complex to introduce targeted double-strand breaks, the CRISPR-Cas system enables the correction of pathogenic mutations and the modulation of gene function with unprecedented efficiency. Its broad applicability spans the correction of inherited genetic defects through homology-directed repair to the disruption of deleterious alleles via non-homologous end joining. In this review, we first outline the molecular architecture and mechanistic basis of CRISPR-Cas9 and then consider its latest applications in modeling, drug screening, small-molecule-mediated editing, and treating hereditary, autoimmune, and oncological diseases. Emphasis is placed on the generation of disease-relevant cellular and animal models and on the potential of CRISPR-Cas9-mediated gene therapy to address hitherto intractable disorders. Finally, we discuss current challenges including off-target activity, gene editing efficiency, delivery constraints, and immunogenicity and highlight emerging strategies to overcome these hurdles and broaden the clinical impact of CRISPR-Cas systems.

Embryonic stem (ES) cells require homologous recombination (HR) to cope with genomic instability caused during self-renewal. Here, we report expression dynamics and localization of endogenous HR factors in DNA break repair of ES cells. In addition, we analyzed gene expression patterns of HR-related factors at the transcript level with RNA-sequencing experiments. We showed that ES cells constitutively expressed diverse HR proteins throughout the cell cycle and that HR protein expression was not significantly changed even in the DNA damaging conditions. We further analyzed that depleting Rad51 resulted in the accumulation of larger single-stranded DNA (ssDNA) gaps, but did not perturb DNA replication, indicating that ES cells were able to enter the G2-phase in the presence of unrepaired DNA gaps, consistent with the possibility that post-replication repair helps avoid stalling at the G2/M checkpoint. Interestingly, caffeine treatment inhibited the formation of Rad51 or Rad54 foci, but not the formation of γH2AX and Exo1 foci, which led to incomplete HR in ssDNA, thus increasing DNA damage sensitivity. Our results suggested that ES cells possess conserved HR-promoting machinery to ensure effective recruitment of the HR proteins to DNA breaks, thereby driving proper chromosome duplication and cell cycle progression in ES cells.

CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.

Cohesin and condensin, two related protein complexes, play essential roles in ensuring the accurate segregation of the genome into daughter cells during cell division. However, the interaction between cohesin and condensin in embryonic stem cells remains unclear, as does the specific function of the meiosis-specific cohesin complex. Cohesin maintains the cohesion of replicated sister chromatids until their separation at anaphase, whereas condensin facilitates the reorganization of chromosomes into a highly compact structure characteristic of mitosis. First, we found via ChIP-seq analysis that cohesins (SMC3, RAD21, and REC8) and condensin (SMC4) share DNA binding sites in close proximity and directly interact with the insulator protein CTCF. Second, siRNA-regulated SMC3 depletion led to nuclear accumulation of SMC4. Third, embryonic stem (ES) cells uniquely harbor cohesin complexes containing the meiotic kleisin subunit REC8. RAD21 knockdown increased the proportion of SMC3–REC8 complexes. Our findings indicate that cohesin and condensin make important contributions to the functions of the chromosomal organization, and that meiotic cohesin may be specifically required for the mitotic program in ES cells.

Young Eun Koh, Eui-Hwan Choi, Jung-Woong Kim, and Keun Pil Kim. Mol. Cells 2022;45:820-32. https://doi.org/10.14348/molcells.2022.2042