Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.

Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genome-wide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.

CHO-Omics Review: The Impact of Current and Emerging Technologies on Chinese Hamster Ovary Based Bioproduction.

CHO cells are the most prevalent platform for modern bio-therapeutic production. Currently, there are several CHO cell lines used in bioproduction with distinct characteristics and unique genotypes and phenotypes. These differences limit advances in productivity and quality that can be achieved by the most common approaches to bioprocess optimization and cell line engineering. Incorporating omics-based approaches into current bioproduction processes will complement traditional methodologies to maximize gains from CHO engineering and bioprocess improvements. In order to highlight the utility of omics technologies in CHO bioproduction, the authors discuss current applications as well as limitations of genomics, transcriptomics, proteomics, metabolomics, lipidomics, fluxomics, glycomics, and multi-omics approaches and the potential they hold for the future of bioproduction. Multiple omics approaches are currently being used to improve CHO bioprocesses; however, the application of these technologies is still limited. As more CHO-omic datasets become available and integrated into systems models, the authors expect significant gains in product yield and quality. While individual omics technologies provide incremental improvements in bioproduction, the authors will likely see the most significant gains by applying multi-omics and systems biology approaches to individual CHO cell lines.

Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion.

There is often interest in dissecting the relative contributions of the N-glycans, O-glycans and glycosphingolipids (GSLs) in regulating complex biological traits like cell signaling, adhesion, development and metastasis. To address this, we developed a CRISPR-Cas9 toolkit to selectively truncate each of these commonly expressed glycan-types. Here, O-glycan biosynthesis was truncated by knocking-out Core 1 ß3Gal-T Specific Molecular Chaperone (COSMC), N-glycans by targeting the ß1,2 GlcNAc-transferase (MGAT1) and GSLs by deleting UDP-glucose ceramide glucosyltransferase (UGCG). These reagents were applied to reveal the glycoconjugates regulating human myeloid cell adhesion to selectins under physiological shear-flow observed during inflammation. These functional studies show that leukocyte rolling on P- and L-selectin is ablated in cells lacking O-glycans, with N-glycan truncation also increasing cell rolling velocity on L-selectin. All three glycan families contributed to E-selectin dependent cell adhesion with N-glycans contributing to all aspects of the leukocyte adhesion cascade, O-glycans only being important during initial recruitment, and GSLs stabilizing slow cell rolling and the transition to firm arrest. Overall, the genome editing tools developed here may be broadly applied in studies of cellular glycosylation.

Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade.

OBJECTIVE: Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion. APPROACH AND RESULTS: A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. CONCLUSIONS: Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes.

The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galß1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.

Entry and killing of Tetrahymena thermophila by bacterially produced Shiga toxin.

UNLABELLED: Phage-encoded Shiga toxin (Stx) acts as a bacterial defense against the eukaryotic predator Tetrahymena thermophila. It is unknown how Stx enters Tetrahymena protozoa or how it kills them. Tetrahymena protozoa are phagocytotic; hence, Stx could gain entry to the cytoplasm through the oral apparatus or via endocytosis. We find that Stx2 can kill T. thermophila protozoa that lack an oral apparatus, indicating that Stx2 can enter these cells via endocytosis. As opposed to the lack of effect on mammalian phagocytes, Stx2 produced by bacteria encapsulated within phagocytotic vesicles is also capable of killing Tetrahymena. Addition of an excess of the carbohydrate binding subunits of Stx2 (StxB) and/or ricin (ricin B) blocks Stx2 cytotoxicity. Thus, regardless of whether Stx2 enters the cytoplasm by endocytosis or from the phagocytotic vesicle, this transport is mediated by a putative glycoconjugate receptor. Bacteriophage-mediated lysis of Stx-encoding bacteria is necessary for Stx toxicity in Tetrahymena; i.e., toxin released as a consequence of digestion of bacteria by Tetrahymena is harmless to the cell. This finding provides a rationale as to why the genes encoding Stx are found almost exclusively on bacteriophages; Stx must be released from the bacteria prior to the digestion of the cell, or it will not be able to exert its cytotoxic effect. It also suggests a reason why other bacterial exotoxins are also found only on temperate bacteriophages. Incubation of Tetrahymena with purified Stx2 decreases total protein synthesis. This finding indicates that, similar to mammalian cells, Stx2 kills Tetrahymena by inactivating its ribosomes. IMPORTANCE: Tetrahymena is a bacterial predator and a model for mammalian phagocytosis and intracellular vesicular trafficking. Phage-encoded exotoxins apparently have evolved for the purpose of bacterial antipredator defense. These exotoxins kill mammalian cells by inactivating universally conserved factors and/or pathways. Tetrahymena and susceptible mammalian cells are killed when exposed to bacteriophage-encoded Shiga toxin (Stx). Stx toxicity in mammalian cells requires Stx binding to the globotriaosyl ceramide (Gb3) receptor, followed by receptor-mediated endocytosis (RME). We show that, similar to mammalian cells, internalized Stx inhibits protein synthesis in Tetrahymena. Although Tetrahymena lacks Gb3, our results suggest that the cytotoxic effect of Stx on Tetrahymena is apparently mediated by a receptor, thereby arguing for the existence of RME in Tetrahymena. As opposed to the case with mammalian phagocytes, Stx produced by bacteria inside Tetrahymena is cytotoxic, suggesting that these cells may represent a "missing link" between unicellular eukaryotic bacterial predators and phagocytotic mammalian cells.

Shiga toxin as a bacterial defense against a eukaryotic predator, Tetrahymena thermophila.

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian "targets," suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that Tetrahymena thermophila, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by Tetrahymena. Bacterially mediated Tetrahymena killing was blocked by mutations that prevented the bacterial SOS response (recA mutations) or by enzymes that breakdown H(2)O(2) (catalase), suggesting that the production of H(2)O(2) by Tetrahymena signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.