CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.

Polypyrimidine tract-binding protein (PTB) is an RNAbinding protein with multiple functions in the regulation of RNA processing and IRES-mediated translation. We report here overexpression of PTB in a majority of epithelial ovarian tumors revealed by immunoblotting and tissue microarray (TMA) staining. By western blotting, we found that PTB was overexpressed in 17 out of 19 ovarian tumor specimens compared to their matchednormal tissues. By TMA staining, we found PTB expression in 38 out of 44 ovarian cancer cases but only in two out of nine normal adjacent tissues. PTB is also overexpressed in SV40 large T-antigen immortalized ovarian epithelial cells compared to normal human ovarian epithelial cells. Using doxycycline-inducible small interfering RNA technology, we found that knockdown of PTB expression in the ovarian tumor cell line A2780 substantially impaired tumor cell proliferation, anchorage-independent growth and in vitro invasiveness. These results suggest that overexpression of PTB is an important component of the multistep process of tumorigenesis, and might be required for the development and maintenance of epithelial ovarian tumors. Moreover, because of its novel role in tumor cell growth and invasiveness, shown here for the first time, PTB may be a novel therapeutic target in the treatment of ovarian cancer.

The breast cancer resistance protein (BCRP) is an ATP-binding cassette half transporter that confers resistance to anticancer drugs such as mitoxantrone, anthracyclines, topotecan, and SN-38. Initial characterization of the BCRP promoter revealed that it is TATA-less with 5 putative Sp1 sites downstream from a putative CpG island and several AP1 sites (K. J. Bailey-Dell et al., Biochim. Biophys. Acta, 1520: 234 -241, 2001). Here, we examined the sequence of the 5-flanking region of the BCRP gene and found a putative estrogen response element (ERE). We showed that estrogen enhanced the expression of BCRP mRNA in the estrogen receptor (ER)-positive T47D:A18 cells and PA-1 cells stably expressing ER␣. In BCRP promoter-luciferase assays, sequential deletions of the BCRP promoter showed that the region between ؊243 and ؊115 is essential for the ER effect. Mutation of the ERE found within this region attenuated the estrogen response, whereas deletion of the site completely abrogated the estrogen effect. Furthermore, electrophoretic mobility shift assays revealed specific binding of ER␣ to the BCRP promoter through the identified ERE. Taken together, we provide evidence herein for a novel ERE in the BCRP promoter.

Tissue repair after spinal cord injury (SCI) requires mobilization of immune and glial cells to form a protective barrier that seals the wound and facilitates debris clearing, inflammatory containment, and matrix compaction. This process involves corralling, wherein phagocytic immune cells become confined to the necrotic core surrounded by an astrocytic border. Here, we elucidate a temporally distinct gene signature in injury-activated microglia/macrophages (IAM), which engages axon guidance pathways. Plexin-B2 is upregulated in IAM, which is required for motosensory recovery after SCI. Plexin-B2 deletion in myeloid cells impairs corralling, leading to diffuse tissue damage, inflammatory spillover, and hampered axon regeneration. Corralling begins early and requires Plexin-B2 in both microglia and macrophages. Mechanistically, Plexin-B2 promotes microglia motility, steers IAM away from colliding cells, and facilitates matrix compaction. Our data thus establish Plexin-B2 as an important link that integrates biochemical cues and physical interactions of IAM with the injury microenvironment during wound healing.