Use Of Mammalian Cells Research Articles

RNA Switches Using Cas Proteins.

Expanding the number of available RNA-binding proteins (RBPs) is vital to establishing posttranscriptional circuits in mammalian cells. We focused on CRISPR-Cas systems and exploited Cas proteins for their versatility as RBPs. The translation of genes encoded in an mRNA becomes regulatable by a Cas protein by inserting a crRNA/sgRNA sequence recognizable by the specific Cas protein into its 5'UTR. These Cas protein-responsive switches vastly expand the available tools in synthetic biology because of the wide range of Cas protein orthologs that can be used as trigger proteins.Here, we describe the design principle of Cas protein-responsive switches, both plasmid and RNA versions, using Streptococcus pyogenes Cas9 (SpCas9) as an example and show an example of its use in mammalian cells, HEK293FT cells.

Impedance In Vitro Assessment for the Detection of Salmonella typhimurium Infection in Intestinal Human Cancer Cells

A significant number of research papers regarding biosensor-related assays for key food safety pathogens based on the use of mammalian cells has been reported. In this study, the Salmonella typhimurium infection progression was monitored in the human colon cancer cell line Caco-2 and the mucus-secreting HT29-MTX-E12, after treatment with five different bacterial MOI for 30 min by comparing the alterations of frequencies recordings with impedance spectroscopy measurements. For this purpose, bacterial adhesion and invasion assays were initially performed. Then, the data obtained from impedance spectroscopy recordings were compared to cell viability data derived from the MTT uptake cell proliferation assay as well as from live cell analysis assays of mitochondrial membrane potential alterations. From our findings a concentration-dependent increase in bacterial colonies occurring from invaded cells was observed upon a higher multiplicity of infection (MOI) bacterial infection at both cell lines. On the contrary, the bacteria infection did not have any impact on the viability of the cells after 1 h of treatment. Differential results were obtained from the measurement of mitochondrial potential at both cell lines. Finally, the impedance values recorded from the 2D, and 3D cultures were concentration-dependent for both cell lines whereas a characteristic pattern specific to each cell line was revealed. Our results indicate that human cell-based bio-electric assays can be a valuable tool for obtaining a unique fingerprint for each bacterial infection in the near future.

Influence of cell specific parameters in a dielectric spectroscopy conversion model used to monitor viable cell density in bioreactors.

In the biopharmaceutical industry, the use of mammalian cells to produce therapeutic proteins is becoming increasingly widespread. Monitoring of these cultures via different analysis techniques is essential to ensure a good quality product while respecting good manufacturing practice (GMP) regulations. PAT (Process Analytical Technologies) tools provide real-time measurements of the physiological state of the culture and enable process automation. Dielectric spectroscopy is a PAT that can be used to monitor the viable cell concentration of living cells (VCC) after processing raw permittivity data. Several modeling approaches exist and estimate biomass with different accuracy. The accuracy of the Cole-Cole and Maxwell Wagner's equations are studied here in the determination of the VCC and cell radius in Chinese hamster ovary (CHO) culture. A sensitivity analysis performed on the parameters entering the equations highlighted the importance of the cell specific parameters such as internal conductivity (σi ) and membrane capacitance (Cm ) in the accuracy of the estimation of VCC and cell radius. The most accurate optimization method found to improve the accuracy involves in-process adjustments of Cm and σi in the model equations with samplings from the bioreactor. This combination of offline and in situ data improved the estimation precision of the viable cell concentration by 69% compared to a purely mechanistic model without offline adjustments. This article is protected by copyright. All rights reserved.

Segmentation strategy of de novo designed four-helical bundles expands protein oligomerization modalities for cell regulation

Protein–protein interactions govern most biological processes. New protein assemblies can be introduced through the fusion of selected proteins with di/oligomerization domains, which interact specifically with their partners but not with other cellular proteins. While four-helical bundle proteins (4HB) have typically been assembled from two segments, each comprising two helices, here we show that they can be efficiently segmented in various ways, expanding the number of combinations generated from a single 4HB. We implement a segmentation strategy of 4HB to design two-, three-, or four-chain combinations for the recruitment of multiple protein components. Different segmentations provide new insight into the role of individual helices for 4HB assembly. We evaluate 4HB segmentations for potential use in mammalian cells for the reconstitution of a protein reporter, transcriptional activation, and inducible 4HB assembly. Furthermore, the implementation of trimerization is demonstrated as a modular chimeric antigen receptor for the recognition of multiple cancer antigens.

Unveiling Cas8 dynamics and regulation of a transposon-encoded cascade-TniQ complex.

Csy (CRISPR-System Yersinia, or ‘Cascade’) is a class 1, type 1F CRISPR-Cas system which utilizes RNA-guided strand invasion to target viral DNA and designate it for cleavage by an external nuclease (Cas2/3). It has been recently shown, in Vibrio Cholerae, that there exists a variant of Cascade encoded in a transposon (Tn6677). This variant binds to a TniQ homodimer, resulting in a ‘Cascade-TniQ’ complex which, unlike the original Cascade that targets viral DNA for cleavage, recruits a heteromeric transposase responsible for integration of Tn6677. This integration process starts with binding of Cascade's crRNA to the DNA target sequence located in the bacterial genome, resulting in the formation of the ‘R Loop,’ this step is the focus of the current research. Both classical molecular dynamics (cMD) and enhanced sampling methods (GaMD) have been employed to better understand the dynamical state of the system in the RNA-only and the DNA-bound configurations. We have found evidence for a multi-state like behavior, with drastic differences in the state population and transition barriers upon DNA binding. Furthermore, it appears that the TniQ homodimer undergoes changes in large-scale conformations dependent on its level of intrinsic stability. This allowed us to make a tentative claim on a role of Zinc-coordination within TniQ's Zinc-finger binding domains. Tying our findings together with data from literature allowed us to make general comments on how the RNA-bound state and R Loop formation may be regulated. Hopefully, these findings will eventually allow for a more streamlined effort in re-engineering Cascade-TniQ for use in mammalian cells.

Exploiting HUH-endonucleases for bioconjugation of mammalian proteins.

The rep domain of HUH-endonucleases process single-stranded DNA (ssDNA) in nature during rolling circle replication, conjugative plasmid transfer, and DNA integration into the host genome by bacteria and viruses. Upon the recognition of a highly conserved nonanucleotide located in a hairpin, HUH-endonucleases catalyze the cleavage reaction of ssDNA, forming a covalent protein-DNA intermediate which has been exploited as a fusion tag for bioconjugation in several biotechnology applications. “HUH-tags” are appealing for their ability to covalently link proteins to DNA in a specific manner and in a ‘one-pot’ reaction which is an inexpensive alternative to other methods that often require the modification of DNA bases or additional steps. To expand the application of HUH-tags for use in mammalian cells and in fusion with mammalian proteins, a panel of HUH-endonucleases from several viral families were characterized for their secretion and catalytic activity in HEK293 cells. Based on robust expression and activity,the WDV rep was used to attach DNA handles to the Notch1 mechanosensing domain for single molecule studies using optical tweezers. Additionally, WDV was expressed with a number of ligands involved in mechanosensing proteins on the surface of cells and the ECM to attach ssDNA for the development of Tension Gauge Tether DNA-based molecular tension sensors.

Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria

Ultrasound allows imaging at a much greater depth than optical methods, but existing genetically encoded acoustic reporters for in vivo cellular imaging have been limited by poor sensitivity, specificity and in vivo expression. Here we describe two acoustic reporter genes (ARGs)—one for use in bacteria and one for use in mammalian cells—identified through a phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea that provide stronger ultrasound contrast, produce non-linear signals distinguishable from background tissue and have stable long-term expression. Compared to their first-generation counterparts, these improved bacterial and mammalian ARGs produce 9-fold and 38-fold stronger non-linear contrast, respectively. Using these new ARGs, we non-invasively imaged in situ tumor colonization and gene expression in tumor-homing therapeutic bacteria, tracked the progression of tumor gene expression and growth in a mouse model of breast cancer, and performed gene-expression-guided needle biopsies of a genetically mosaic tumor, demonstrating non-invasive access to dynamic biological processes at centimeter depth.

The Next Frontier of 3D Bioprinting: Bioactive Materials Functionalized by Bacteria.

3D bioprinting has become a flexible technical means used in many fields. Currently, research on 3D bioprinting is mainly focused on the use of mammalian cells to print organ and tissue models, which has greatly promoted progress in the fields of tissue engineering, regenerative medicine, and pharmaceuticals. In recent years, bacterial bioprinting has gradually become a rapidly developing research fields, with a wide range of potential applications in basic research, biomedicine, bioremediation, and other field. Here, this works reviews new research on bacterial bioprinting, and discuss its future research direction.

Production of Eukaryotic Glycoproteins for Structural and Functional Studies Using Expi293F Cells.

Milligram quantities of pure proteins are required for structural, functional, and pharmaceutical screening studies. These requirements can be challenging for a majority of important therapeutic targets that are secreted glycoproteins, receptors, membrane proteins, or large cytosolic complexes. Here, we present protocols for producing and purifying large amounts of secreted glycoproteins using the mammalian cell-based Expi293F system via large-scale transient transfection. This system can be easily adapted for the production of membrane proteins and large cytosolic complexes. The method can be utilized to quickly evaluate numerous expression constructs to identify optimal expressers. Use of mammalian cells ensures proper post-translational modifications, including disulfide bonds and glycosylation, that can be important for accurate functional studies. In addition, minor modifications can be introduced to produce labeled or deglycosylated proteins for structural studies by X-ray crystallography, nuclear magnetic resonance, or cryo-electron microscopy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Production of milligram quantities of plasmid DNA for large-scale transient transfection Basic Protocol 2: Large-scale culture and transient transfection of Expi293F cells Basic Protocol 3: Purification of hexahistidine-tagged proteins from medium.

QS66. The Safety of Elution of Multiple Antibiotics From Collagen-rich Hydrogel for Topical Treatment of Chronic Wounds

Purpose: Chronic wounds pose a significant challenge to modern healthcare systems, affecting 1-2% of the population in developed countries with costs ranging between $28.1 and $96.8 billion annually. Overtime chronic wounds tend to become colonized or infected with multiple different bacteria resulting in patients being treated with several rounds of systemic oral and IV antibiotics. Subsequently, antibiotic resistance develops, and wounds become harder to treat. Comorbidities, such as diabetes and peripheral vascular disease, also make treatment more challenging. Ideally chronic wounds would be treated topically with customized antimicrobial combinations unique to each patient’s wound.In vivo, we have previously shown that a topical collagen-rich hydrogel (cHG) can provide controlled elution of single antibiotics leading to inhibition of bacterial growth while avoiding cytotoxicity towards host cells in a stented wound model. We hypothesize that the elution of multiple antibiotics from collagen-rich hydrogel will also be well-tolerated in mammalian cells. Methods: Antibiotic Selection: We performed a literature search to determine the antibiograms bacteria isolated from chronic diabetic wounds; the most common microbes were Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus spp., and beta-hemolytic streptococci. This informed our choice of antibiotics. We tested four antibiotic combinations from six antibiotics. While there are 15 two-antibiotic permutations, these combinations provided the most extensive coverage. Collagen hydrogel formation: 2.5% collagen-rich thermoresponsive hydrogel (cHG) was synthesized according to a previously established protocol. After confirmation of composition and gelation, cHG-antibiotic solutions were prepared by determining 100x the minimum inhibitory concentration of each antibiotic and adjusting the final volume of hydrogel to 100 mL per well. The antibiotic concentrations used were 400 ug/mL vancomycin, 400 ug/mL imipenem, 200 ug/mL ciprofloxacin, 500 ug/mL ceftazidime, 1.0 mg/mL gentamicin, and 100 ug/mL clindamycin.Mammalian cell cytotoxicity: Human adipose-derived stem cells (ASCs), mouse ASCs, human fibroblasts (FBs), and mouse FBs were used to study cytotoxicity in both differentiated and undifferentiated mammalian cells. Cells were seeded onto 24-well culture plates at a density of 2 × 104 cells/ml, allowed to attach overnight, and treated with cHG+antibiotic, incubated until 24, 48, and 72-hour time points.A live/dead viability/cytotoxicity assay was used according to the manufacturer’s instructions, and images were analyzed using MATLAB. All experiments were performed in triplicates. Results: We observed no significant cell death in differentiated and undifferentiated cells in both mouse and human cell lines treated with antibiotic-eluting hydrogel with multiple combinations of antibiotics. All cell lines had >90% survival at all timepoints. Conclusion: This study demonstrates that the application of a topical drug-eluting hydrogel with a combination of antibiotics in the hydrogel is safe for use in mammalian cells. This could transform the treatment of chronic diabetic wounds, which are often polymicrobial with unique resistance patterns requiring multiple antibiotics.

Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active

Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.

Glycosylase base editors enable C-to-A and C-to-G base changes.

Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.

A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast

The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.

Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production.

The production of virus-like particles (VLPs) has gained importance over the last few years owing to the benefits they provide compared to conventional vaccines. The biopharmaceutical industry is currently searching for safer candidates based on VLPs for new and existing vaccines and implementing new methods of manufacturing, thus allowing a more sustainable, effective, and species-specific production. Despite achieving lower yields compared to traditional platforms, the use of mammalian cells provides the right post-translational modifications, and consequently, the intensification of bioprocesses using mammalian cell platforms has become a matter of pressing concern. One of the methods subjected to intensification is transient gene expression, which has been proven to be highly effective regarding VLP production for preclinical or even clinical trials. In this work, a multiplexed quantitative proteomic approach has been applied to study the molecular characteristics of HEK293 cell cultures when growing at cell densities higher than 4 × 106 cells/mL and to study the effects related to cell transfection and VLP production. The obtained results revealed a set of functional and metabolic profiles of HEK293 under these three different conditions that allowed the identification of physiological bottlenecks regarding VLP production. Regarding the cell density effect, molecular alterations in the cell biology were proposed to help explain the difficulty for the cells to be transfected at higher densities. In addition, an overall disruption of cellular homeostasis after transfection was observed based on altered biological processes, and after identifying potential pathways liable to be optimized via metabolic engineering, different solutions were proposed to improve VLP production.

Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology.

The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.

Biogenic Aspergillus tubingensis silver nanoparticles' in vitro effects on human umbilical vein endothelial cells, normal human fibroblasts, HEPG2, and Galleria mellonella.

Silver nanoparticles (AgNPs) are widely incorporated into different hygiene, personal care, and healthcare products. However, few studies have been undertaken to determine the effects of biogenic AgNPs on human health. The effect of biosynthesized AgNPs using the fungus Aspergillus tubingensis culture was evaluated on human umbilical vein endothelial cells (HUVECs), normal human fibroblasts (FN1), human hepatoma cells (HEPG2) and a Galleria mellonella model. HUVECs were more susceptible to biogenic AgNPs than normal fibroblasts FN1 and intense cytotoxicity was observed only for very high concentrations at and above 2.5 μM for both cells. Normal human fibroblasts FN1 exposed to AgNPs for 24 h showed viability of 98.83 ± 8.40% and 94.86 ± 5.50% for 1.25 and 2.5 μM, respectively. At 5 and 10 μM, related to the control, an increase in cell viability was observed being 112.66 ± 9.94% and 117.86 ± 8.86%, respectively. Similar results were obtained for treatment for 48 and 72 h. At 1.25, 2.5, 5 and 10 μM of AgNPs, at 24 h, HUVECs showed 51.34 ± 7.47%, 27.01 ± 5.77%, 26.00 ± 3.03% and 27.64 ± 5.85% of viability, respectively. No alteration in cell distribution among different cycle phases was observed after HUVEC and normal fibroblast FN1 exposure to AgNPs from 0.01 to 1 μM for 24, 48 and 72 h. Based on the clonogenic assay, nanoparticles successfully inhibited HEPG2 cell proliferation when exposed to concentrations up to 1 μM. In addition to that, AgNPs did not induce senescence and no morphological alteration was observed by scanning electron microscopy on the endothelial cells. In the larvae of the wax moth, Galleria mellonella, a model for toxicity, AgNPs showed no significant effects, which corroborates to the safety of their use in mammalian cells. These results demonstrate that the use of A. tubingensis AgNPs is a promising biotechnological approach and these AgNPs can be applied in several biomedical situations.

Synthetic Biology: Engineering Mammalian Cells To Control Cell-to-Cell Communication at Will.

Cell-to-cell communication plays a key role in the regulation of many natural biological processes. Recent advances in mammalian synthetic biology are making it possible to rationally engineer cell-to-cell communication for therapeutic and other purposes. Here, we review state-of-the-art engineering principles to control cell-to-cell communication, focusing on communication between mammalian cells with diffusible factors (e.g., small molecules or exosomes) or direct cell contact, and on interkingdom communication between mammalian cells and bacteria. Potential applications include construction of artificial tissues able to perform complex computations, sophisticated cell-based cancer therapies, use of mammalian cells as a new class of cargo delivery modality, development of design principles to control pattern formation of cell populations, and treatment of infectious diseases. We also discuss the challenges facing practical applications, and possible enabling technologies to overcome them.

Cancer cells biomineralize ionic gold into nanoparticles-microplates via secreting defense proteins with specific gold-binding peptides

Cancer cells have the capacity to synthesize nanoparticles (NPs). The detailed mechanism of this process is not very well documented. We report the mechanism of biomineralization of aqueous gold chloride into NPs and microplates in the breast-cancer cell line MCF7. Spherical gold NPs are synthesized in these cells in the presence of serum in the culture media by the reduction of HAuCl4. In the absence of serum, the cells exhibit gold microplate formation through seed-mediate growth albeit slower reduction. The structural characteristics of the two types of NPs under different media conditions were confirmed using scanning electron microscopy (SEM); crystallinity and metallic properties were assessed with transmission electron microscopy (TEM) and x-ray photoelectron spectroscopy (XPS). Gold-reducing proteins, related to cell stress initiate the biomineralization of HAuCl4 in cells (under serum free conditions) as confirmed by infrared (IR) spectroscopy. MCF7 cells undergo irreversible replicative senescence when exposed to a high concentration of ionic gold and conversely remain in a dormant reversible quiescent state when exposed to a low gold concentration. The latter cellular state was achievable in the presence of the rho/ROCK inhibitor Y-27632. Proteomic analysis revealed consistent expression of specific proteins under serum and serum-free conditions. A high-throughput proteomic approach to screen gold-reducing proteins and peptide sequences was utilized and validated by quartz crystal microbalance with dissipation (QCM-D). Statement of significanceCancer cells are known to synthesize gold nanoparticles and microstructures, which are promising for bioimaging and other therapeutic applications. However, the detailed mechanism of such biomineralization process is not well understood yet. Herein, we demonstrate that cancer cells exposed to gold ions (grown in serum/serum-free conditions) secrete shock and stress-related proteins with specific gold-binding/reducing polypeptides. Cells undergo reversible senescence and can recover normal physiology when treated with the senescence inhibitor depending on culture condition. The use of mammalian cells as microincubators for synthesis of such particles could have potential influence on their uptake and biocompatibility. This study has important implications for in-situ reduction of ionic gold to anisotropic micro-nanostructures that could be used in-vivo clinical applications and tumor photothermal therapy.

Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry.

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.