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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a versatile technology that allows precise modification of genes. One of its most promising applications is in cancer treatment. By targeting and editing specific genes involved in cancer development and progression, CRISPR has the potential to become a powerful tool in the fight against cancer. This review aims to assess the recent progress in CRISPR technology for cancer research and to examine the obstacles and potential strategies to address them. The two most commonly used CRISPR systems for gene editing are CRISPR/Cas9 and CRISPR/Cas12a. CRISPR/Cas9 employs different repairing systems, including homologous recombination (HR) and nonhomologous end joining (NHEJ), to introduce precise modifications to the target genes. However, off-target effects and low editing efficiency are some of the main challenges associated with this technology. To overcome these issues, researchers are exploring new delivery methods and developing CRISPR/Cas systems with improved specificity. Moreover, there are ethical concerns surrounding using CRISPR in gene editing, including the potential for unintended consequences and the creation of genetically modified organisms. It is important to address these issues through rigorous testing and strict regulations. Despite these challenges, the potential benefits of CRISPR in cancer therapy cannot be overlooked. By introducing precise modifications to cancer cells, CRISPR could offer a targeted and effective treatment option for patients with different types of cancer. Further investigation and development of CRISPR technology are necessary to overcome the existing challenges and harness its full potential in cancer therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

MicroRNAs profoundly impact hematopoietic cells by regulating progenitor cell-fate decisions, as well as mature immune effector function. However to date, microRNAs that regulate hematopoietic stem cell (HSC) function have been less well characterized. Here we show that microRNA-125b (miR-125b) is highly expressed in HSCs and its expression decreases in committed progenitors. Overexpression of miR-125b in mouse HSC enhances their function, demonstrated through serial transplantation of highly purified HSC, and enriches for the previously described Slamf1(lo)CD34(-) lymphoid-balanced and the Slamf1(neg)CD34(-) lymphoid-biased cell subsets within the multipotent HSC (CD34-KLS) fraction. Mature peripheral blood cells derived from the miR-125b-overexpressing HSC are skewed toward the lymphoid lineage. Consistent with this observation, miR-125b overexpression significantly increases the number of early B-progenitor cells within the spleen and induces the expansion and enrichment of the lymphoid-balanced and lymphoid-biased HSC subset via an antiapoptotic mechanism, reducing the mRNA expression levels of two proapoptotic targets, Bmf and KLF13. The antiapoptotic effect of miR-125b is more pronounced in the lymphoid-biased HSC subset because of their intrinsic higher baseline levels of apoptosis. These effects of miR-125b are associated with the development of lymphoproliferative disease, marked by expansion of CD8(+) T lymphocytes. Taken together, these data reveal that miR-125b regulates HSC survival and can promote lymphoid-fate decisions at the level of the HSC by preferentially expanding lymphoid-balanced and lymphoid-biased HSC.

Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that approximately 1-5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12-14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1(-/-) mice do not exhibit severe hematopoietic defects; however, Esam1(-/-) BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1(-/-) mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506- and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (DeltaDeltaG(U-F)) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.