Katie Chan

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

Interleukin (IL)-17 is a recently discovered cytokine, which is proposed to play a role in neutrophilic airway inflammation via the release of proinflammatory cytokines and chemokines. To evaluate the role of IL-17 in inflammatory protein production from the airway epithelium, we have analyzed the effects of IL-17 on primary human bronchial epithelial cells (HBECs). Using gene arrays, changes in gene expression in response to IL-17 stimulation were investigated and only IL-8, growth-related oncogene (Gro)alpha, and granulocyte colony-stimulating factor (G-CSF) were found to be upregulated. Secretion of IL-8, Groalpha, and G-CSF in response to IL-17 was measured in HBEC cell culture supernatants by enzyme-linked immunosorbent assay. Upregulation of Groalpha, IL-8, and G-CSF was observed to be 8-, 5-, and 8-fold, respectively, after 48 h stimulation with IL-17. When tested at equivalent concentrations, IL-17 was found to be 2- to 3-fold more potent than tumor necrosis factor (TNF)-alpha in stimulating release of Groalpha and G-CSF from HBECs. In addition, IL-17 was found to synergistically enhance TNF-alpha-induced production of IL-8, Groalpha, and G-CSF. It is proposed that IL-17 may play an important role in neutrophil recruitment via stimulating the release of IL-8, Groalpha, and G-CSF from airway epithelial cells.

Mitochondria play a critical role in generating most of the cell's energy as ATP. They are also involved in other metabolic processes such as urea generation, haem synthesis and fatty acid beta-oxidation. Disruption of mitochondrial function by drugs can result in cell death by necrosis or can signal cell death by apoptosis (e.g., following cytochrome c release). Drugs that injure mitochondria usually do so by inhibiting respiratory complexes of the electron chain; inhibiting or uncoupling oxidative phosphorylation; inducing mitochondrial oxidative stress; or inhibiting DNA replication, transcription or translation. It is important to test for mitochondrial toxicity early in drug development as impairment of mitochondrial function can induce various pathological conditions that are life threatening or can increase the progression of existing mitochondrial diseases.

Restriction endonuclease analysis (REA) was performed on the total cellular DNA from each of 396 strains of mutans streptococci (1) to determine its potential usefulness for the study of transmission of the organism and (2) to document the proportions and variety of strains harbored by members of a small group of families. The DNA was digested with restriction enzyme EcoRI and/or HindIII, electrophoresed on agarose gels, and the resulting patterns compared. The strains examined included fresh isolates from 58 subjects, including 19 strains from each member of five families. The sensitivity and reproducibility of REA patterns from the mutans streptococci seemed ideal for studies of their epidemiology and transmission. The pattern of each isolate from humans was unique, except for isolates from the same individual or from the same family. REA types from subjects from different families were always heterogeneous. A high frequency of multiple REA types (up to 5) was observed in many subjects. While evidence for intra-familial transmission was obtained, including transmission between spouses, there was also strong evidence of frequent sources of infection outside of the family. Mutations of strains to streptomycin resistance or to lactate dehydrogenase deficiency caused no detectable change in the REA patterns. The lack of plasmids in any of the 57 fresh isolates that were examined for them suggested that they may have contributed little to the heterogeneity of the patterns seen.