Combinatorial Phenotypes Research Articles

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here, we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results with those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides.

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance, and epiphytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these pathways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival, and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we analysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT–PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature, and surface association strongly affect the expression of different polysaccharides under the control of the signalling protein genes ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides play a role in mediating leaf adhesion, while α-glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection.

A novel immature natural killer cell subpopulation predicts relapse after cord blood transplantation

AbstractNatural killer (NK) cells are highly heterogeneous, with vast phenotypic and functional diversity at the single-cell level. They are involved in the innate immune response against malignant and virus-infected cells. To understand the effect of NK diversity during immune recovery on the antitumor response after cord blood transplantation (CBT), we used high-dimensional mass cytometry and the metrics of NK cell diversity to study the NK cell repertoire in serial samples from 43 CBT recipients. A higher-diversity index based on single-cell combinatorial phenotypes was significantly associated with a lower risk for relapse after CBT (P = .005). Cytomegalovirus reactivation was a major factor in the development of a more diverse NK repertoire after CBT. Notably, we identified a group of patients whose CB-derived NK cells after transplantation possessed an immature phenotype (CB-NKim), characterized by poor effector function and a low diversity index. Frequencies of CB-NKim of 11.8% or higher during the early post-CBT recovery phase were highly predictive for relapse (area under the curve [AUC], 0.979), a finding that was validated in a second independent cohort of patients (n = 25; AUC, 0.977). Moreover, we showed that the maturation, diversity, and acquisition of effector function by CB-NKim early after CBT were driven by interleukin 15. Our data indicate that the diversity of the NK cell repertoire after CBT contributes importantly to the risk for subsequent relapse. We suggest that the use of diversity metrics and high-dimensional mass cytometry may be useful tools in predicting clinical outcomes and informing the design of therapeutic strategies to prevent relapse after CBT.

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current approaches suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from S. aureus and S. pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The “Big Papi” (Paired aureus and pyogenes for intereactions) approach described here will be widely applicable for the study of combinatorial phenotypes.

Pregnancy probabilistically augments potential precursors to chronic, immune-mediated or autoimmune lacrimal gland infiltrates

PurposeThis study asked whether pregnancy, a risk factor for dry eye disease associated with both chronic, immune-mediated- and autoimmune etiologies, augments development of clusters of coordinately functioning cells (CCFC) that may be precursors to pathological lacrimal gland infiltrates. MethodsLacrimal glands were from six virgin- and six term-pregnant rabbits of the same age and environmental exposure history. Seventy-two immune response-related gene transcripts were assayed by real time RT-PCR. Principal component (PC) analysis identified transcript signatures of CCFC contributing negative (⊖) or positive (⊕) PC loadings and determined gland PC projections, which reflect levels of CCFC development. ResultsThree CCFC were of interest as potential precursors to pathological infiltrates. CCFC 1⊖ was suggestive of an ectopic lymphoid structure with resting T cells and B cells. CCFC 1⊕ was suggestive of an immune-mediated infiltrate with TH1 cells and mature, cytotoxic B cells. CCFC 2⊖ was suggestive of an ectopic lymphoid structure with activated T cells, mature B cells, germinal center, and plasmacytes. CCFC 4⊖ and CCFC 5⊖ also included plasmacytes. Pregnancy augmented CCFC 1⊖ in some glands; augmented CCFC 1⊕ in others; and augmented CCFC 2⊖, CCFC 4⊖, and CCFC 5⊖ different combinations. ConclusionsPotential precursors of pathological infiltrates form in the lacrimal glands by the time of sexual maturity. Pregnancy augments lacrimal gland plasmacyte populations, and it can augment development of potential precursors to either chronic, immune-mediated infiltrates or autoimmune infiltrates of various phenotypes. Systemic and strictly local, probabilistic phenomena interact with pregnancy to determine which combinatorial phenotypes are favored.

ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC.

BackgroundWe have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized.MethodsWe overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients.ResultsOverexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 and c-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients’ outcome was independent of tumor grade, stage and size, and ER status.ConclusionADA3 overexpression enhances cell proliferation that is associated with increased expression of c-MYC. Expression patterns with respect to ADA3/c-MYC can divide patients into four significantly different subgroups, with c-MYC High and ADA3 Low status independently predicting poor survival in patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0770-9) contains supplementary material, which is available to authorized users.

A combinatorial radiographic phenotype may stratify patient survival and be associated with invasion and proliferation characteristics in glioblastoma.

Individual MRI characteristics (e.g., volume) are routinely used to identify survival-associated phenotypes for glioblastoma (GBM). This study investigated whether combinations of MRI features can also stratify survival. Furthermore, the molecular differences between phenotype-induced groups were investigated. Ninety-two patients with imaging, molecular, and survival data from the TCGA (The Cancer Genome Atlas)-GBM collection were included in this study. For combinatorial phenotype analysis, hierarchical clustering was used. Groups were defined based on a cutpoint obtained via tree-based partitioning. Furthermore, differential expression analysis of microRNA (miRNA) and mRNA expression data was performed using GenePattern Suite. Functional analysis of the resulting genes and miRNAs was performed using Ingenuity Pathway Analysis. Pathway analysis was performed using Gene Set Enrichment Analysis. Clustering analysis reveals that image-based grouping of the patients is driven by 3 features: volume-class, hemorrhage, and T1/FLAIR-envelope ratio. A combination of these features stratifies survival in a statistically significant manner. A cutpoint analysis yields a significant survival difference in the training set (median survival difference: 12 months, p = 0.004) as well as a validation set (p = 0.0001). Specifically, a low value for any of these 3 features indicates favorable survival characteristics. Differential expression analysis between cutpoint-induced groups suggests that several immune-associated (natural killer cell activity, T-cell lymphocyte differentiation) and metabolism-associated (mitochondrial activity, oxidative phosphorylation) pathways underlie the transition of this phenotype. Integrating data for mRNA and miRNA suggests the roles of several genes regulating proliferation and invasion. A 3-way combination of MRI phenotypes may be capable of stratifying survival in GBM. Examination of molecular processes associated with groups created by this combinatorial phenotype suggests the role of biological processes associated with growth and invasion characteristics.

Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis.

Clinicopathologic and molecular significance of phospho-Akt expression in early invasive breast cancer

Akt/PKB serine/threonine kinase is a leading signalling modulator for several cellular processes including metabolism, growth, proliferation and survival. However, complexity and diversity in the upstream/downstream arms of Akt pathway, as recent genetic studies reported, challenge considerably the evolvement of effective targeted therapies. The aim was to study the expression of phospho-Akt1 (pAkt) in breast cancer (BC), with respect to different component proteins upstream/downstream of Akt pathway activation, clinicopathologic parameters and patients' outcome. pAkt (Ser473) was evaluated by immunohistochemistry, on tissue microarrays containing 1,202 early invasive BC with long-term clinical follow-up. Seventy-six percent of the studied tumours overexpressed pAkt, where it was associated with expression of oestrogen and androgen receptors, PIK3CA, cytokeratin (CK)18, CK19 and PTEN. Loss of pAkt was correlated with high grade, CK5/6, p53 and high Ki67 labelling index. Higher proportions of luminal tumours were pAkt positive relative to triple negative/basal subtypes. However, pAkt overexpression was not associated with breast cancer specific (BCSS) or metastasis-free survival (MFS). Four tumour phenotypes were identified based on PIK3CA and pAkt expression, with substantial proportions being PIK3CA⁻/pAkt⁺ or PIK3CA⁺/pAkt⁻. These four combinatorial phenotypes were significantly associated with BCSS (p = 0.001) and MFS (p = 0.002). Although pAKT is an oncogene correlated with poor prognostic variables, it was not a prognostic marker. Combinatorial phenotypic groups of PIK3CA/pAkt denoted functional complexity, at translational level, within the upstream and downstream arms of Akt activation with significant impact on patients' outcome. These findings may help development more adequate therapeutic regimens for specific subgroups of this key cancer pathway.