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Cytosine and adenine base editor ribonucleoproteins show precise base editing with reduced DNA and RNA off-target effects.

When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a gene-editing technology. Nanoparticle delivery systems have attracted attention because of the limitations of conventional viral vectors. In this review, we assess the efficiency of various nanoparticles, including lipid-based, polymer-based, inorganic, and extracellular vesicle-based systems, as non-viral vectors for CRISPR/Cas9 delivery. We discuss their advantages, limitations, and current challenges. By summarizing recent advancements and highlighting key strategies, this review aims to provide a comprehensive overview of the role of non-viral delivery systems in advancing CRISPR/Cas9 technology for clinical applications and gene therapy.

Bone morphogenetic protein-2 (BMP-2) is known to induce both osteogenic and chondrogenic commitment of human mesenchymal stem cells (hMSCs). However, factors influencing BMP-2-dependent chondrogenic and osteogenic differentiation have not been investigated. In this study, we demonstrated that extracellular microenvironments, in the form of cell-derived matrices, play important roles in determining the specific lineage commitment of hMSCs in the presence of BMP-2. Extracellular matrices (ECMs) derived from osteoblasts and chondrocytes were utilized to regulate cell differentiation. Osteogenic and chondrogenic differentiation of hMSCs cultured on the two different cell-derived ECMs were assessed by quantitative real-time-polymerase chain reaction, immunocytochemistry, and western blot analysis. To minimize the effects of the cell-adhesion proteins contained in serum on the ECMs, hMSCs were cultured in serum-free osteogenic or chondrogenic differentiation medium. Fibronectin-, collagen type I-, or collagen type II-coated substrates were utilized as ECM controls. The ECM specific to each cell type promoted lineage-specific commitment of hMSCs in the presence of BMP-2, that is, osteoblast- and chondrocyte-derived ECM promoted osteogenic and chondrogenic commitment, respectively. Therefore, cell-specific ECMs are capable of modulating the BMP-2-induced osteogenic and chondrogenic differentiation of hMSCs.