Guidelines for optimized gene knockout using CRISPR/Cas9.

CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.

Single-molecule DNA-mapping and whole-genome sequencing of individual cells.

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

Automating ChIP-seq experiments to generate epigenetic profiles on 10,000 HeLa cells.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

MicroRNA-21 exhibits antiangiogenic function by targeting RhoB expression in endothelial cells.

BACKGROUND: MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated. METHODOLOGY/PRINCIPAL FINDINGS: We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization. CONCLUSIONS/SIGNIFICANCE: Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

Sprouty1, a new target of the angiostatic agent 16K prolactin, negatively regulates angiogenesis.

BACKGROUND: Disorganized angiogenesis is associated with several pathologies, including cancer. The identification of new genes that control tumor neovascularization can provide novel insights for future anti-cancer therapies. Sprouty1 (SPRY1), an inhibitor of the MAPK pathway, might be one of these new genes. We identified SPRY1 by comparing the transcriptomes of untreated endothelial cells with those of endothelial cells treated by the angiostatic agent 16 K prolactin (16 K hPRL). In the present study, we aimed to explore the potential function of SPRY1 in angiogenesis. RESULTS: We confirmed 16 K hPRL induced up-regulation of SPRY1 in primary endothelial cells. In addition, we demonstrated the positive SPRY1 regulation in a chimeric mouse model of human colon carcinoma in which 16 K hPRL treatment was shown to delay tumor growth. Expression profiling by qRT-PCR with species-specific primers revealed that induction of SPRY1 expression by 16 K hPRL occurs only in the (murine) endothelial compartment and not in the (human) tumor compartment. The regulation of SPRY1 expression was NF-κB dependent. Partial SPRY1 knockdown by RNA interference protected endothelial cells from apoptosis as well as increased endothelial cell proliferation, migration, capillary network formation, and adhesion to extracellular matrix proteins. SPRY1 knockdown was also shown to affect the expression of cyclinD1 and p21 both involved in cell-cycle regulation. These findings are discussed in relation to the role of SPRY1 as an inhibitor of ERK/MAPK signaling and to a possible explanation of its effect on cell proliferation. CONCLUSIONS: Taken together, these results suggest that SPRY1 is an endogenous angiogenesis inhibitor.

Antiangiogenic liposomal gene therapy with 16K human prolactin efficiently reduces tumor growth.

Human 16K PRL (16K hPRL) is a potent inhibitor of angiogenesis both in vitro and in vivo. It has been shown to prevent tumor growth in three xenograft mouse models. Here we have used a gene transfer method based on cationic liposomes to produce 16K hPRL and demonstrate that 16K hPRL inhibits tumor growth in a subcutaneous B16F10 mouse melanoma model. Computer-assisted image analysis shows that 16K hPRL treatment results in the reduction of tumor vessel length and width, leading to a 57% reduction in average vessel size. We thus show, for the first time, that administration of the 16K hPRL gene complexed to cationic liposomes is effective to maintain antiangiogenic activities of 16K hPRL level.

The angiostatic 16K human prolactin overcomes endothelial cell anergy and promotes leukocyte infiltration via nuclear factor-kappaB activation.

The 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent angiostatic factor that inhibits tumor growth in mouse models. Using microarray experiments, we have dissected how the endothelial-cell genome responds to 16K hPRL treatment. We found 216 genes that show regulation by 16K hPRL, of which a large proportion turned out to be associated with the process of immunity. 16K hPRL induces expression of various chemokines and endothelial adhesion molecules. These expressions, under the control of nuclear factor-kappaB, result in an enhanced leukocyte-endothelial cell interaction. Furthermore, analysis of B16-F10 tumor tissues reveals a higher expression of adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, or E-selectin) in endothelial cells and a significantly higher number of infiltrated leukocytes within the tumor treated with 16K hPRL compared with the untreated ones. In conclusion, this study describes a new antitumor mechanism of 16K hPRL. Because cellular immunity against tumor cells is a crucial step in therapy, the discovery that treatment with 16K hPRL overcomes tumor-induced anergy may become important for therapeutic perspectives.