Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700–2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>

Are humanized IgE reporter systems potential game changers in serological diagnosis of human parasitic infection?

Immunoglobulin E (IgE) is thought to have evolved to protect mammalian hosts against parasitic infections or toxins and plays a central role in the pathogenesis, diagnosis, and therapy of IgE-mediated allergy. Despite the prominence of IgE responses in most parasitic infections, and in stark contrast to its use in the diagnosis of allergy, this isotype is almost completely unexploited for parasite diagnosis. Here, we discuss the perceived or real limitations of IgE-based diagnosis in parasitology and suggest that the recent creation of a new generation of very sensitive cellular IgE-based reporters may represent a powerful new diagnostic platform, but needs to be based on a very careful choice of diagnostic allergens.

Selective destabilization of polypeptides synthesized from NMD-targeted transcripts

The translation of mRNAs that contain a premature termination codon (PTC) generates truncated proteins that may have toxic dominant negative effects. Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that degrades PTC-containing mRNAs to limit the production of truncated proteins. NMD activation requires a ribosome terminating translation at a PTC, but what happens to the polypeptides synthesized during the translation cycle needed to activate NMD is incompletely understood. Here, by establishing reporter systems that encode the same polypeptide sequence before a normal or premature termination codon, we show that termination of protein synthesis at a PTC is sufficient to selectively destabilize polypeptides in mammalian cells. Proteasome inhibition specifically rescues the levels of nascent polypeptides produced from PTC-containing mRNAs within an hour, but also disrupts mRNA homeostasis within a few hours. PTC-terminated polypeptide destabilization is also alleviated by depleting the central NMD factor UPF1 or SMG1, the kinase that phosphorylates UPF1 to activate NMD, but not by inhibiting SMG1 kinase activity. Our results suggest that polypeptide degradation is linked to PTC recognition in mammalian cells and clarify a framework to investigate these mechanisms.

Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria

Purpose In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter. Procedures In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR. Results In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division. Conclusions Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

The Endogenous RIG-I Ligand Is Generated in Influenza A-Virus Infected Cells

As a result of a viral infection, viral genomes are not only recognized by RIG-I, but also lead to the activation of RNase L, which cleaves cellular RNA to generate the endogenous RIG-I ligand (eRL). The eRL was previously identified as a specific sequence derived from the internal transcribed spacer region 2, which bears a 2′3′ cyclic phosphate instead of the common 5′ triphosphate. By now, the generation of the eRL and its immunostimulatory effect were shown both in vitro and in reporter systems. In this work, we aimed to elucidate whether the eRL is also generated in Influenza A (IAV) and vesicular stomatitis virus (VSV) infected cells. RNA was extracted from virus-infected cells and used for immunostimulations as well as specific PCR-strategies to detect eRL cleavage. We show that the eRL is generated in IAV infected HEK293 cells, but we could not detect specific eRL fragments in VSV infected cells. Further, RIG-I mediated IFN-response depends not only on viral genomes but also on the eRL, as immunostimulatory properties remain present under 5′triphosphate degrading conditions. In summary, we prove the IAV infection induced eRL generation in HEK293 cells, amplifying the innate immune response.

Differential Expression of Yersinia pseudotuberculosis General Porin Genes during Short- and Long-Term Antibiotic Stresses

Here, we investigated general porin regulation in Yersinia pseudotuberculosis 488, the causative agent of Far Eastern scarlet-like fever, in response to sublethal concentrations of antibiotics. We chose four antibiotics of different classes and measured gene expression using qRT-PCR and GFP reporter systems. Our data showed temporal regulation of the general porin genes ompF and ompC caused by antibiotic stress. The porin transcription initially decreased, providing early defensive response of the bacterium, while it returned to that of the untreated cells on prolonged antibiotic exposure. Unlike the major porin genes, the transcription of the alternative porin genes ompX and lamB was increased. Moreover, a short-term ompR- and marA-mediated porin regulation was observed. The main finding was a phenotypic heterogeneity of Y. pseudotuberculosis population manifested in variable porin gene expression under carbenicillin exposure. This may offer adaptive fitness advantages for a particular bacterial subpopulation.

Tools, strains, and strategies to effectively conduct anaerobic and aerobic transcriptional reporter screens and assays in Staphylococcus aureus

Transcriptional reporters are reliable and time-tested tools to study gene regulation. In Staphylococcus aureus, β-galactosidase (lacZ)-based genetic screens are not widely used because of the necessity of selectable markers for strain construction and the production of staphyloxanthin pigment which obfuscates results. We describe a series of vectors that allow for markerless insertion of codon-optimized lacZ-based transcriptional reporters. The vectors encode for different ribosomal binding sites allowing for tailored lacZ expression. A ΔcrtM::kanR deletion insertion mutant was constructed that prevents the synthesis of staphyloxanthin, thereby permitting blue-white screening without the interference of carotenoid production. We demonstrate the utility of these vectors to monitor aerobic and anaerobic transcriptional activity. For the latter, we describe the use of a ferrocyanide-ferricyanide redox system (Fe(CN)63–/4–) permitting blue-white screening in the absence of oxygen. We also describe additional reporter systems and methods for monitoring transcriptional activity during anaerobic culture including a FAD-binding fluorescent protein (EcFbFP), alpha-hemolysin (hla), or lipase (geh). The systems and methods described are compatible with vectors utilized to create and screen high-density transposon mutant libraries.