Mammalian Pathway Research Articles

Cis-Regulatory Elements in Mammals.

In cis-regulatory elements, enhancers and promoters with complex molecular interactions are used to coordinate gene transcription through physical proximity and chemical modifications. These processes subsequently influence the phenotypic characteristics of an organism. An in-depth exploration of enhancers and promoters can substantially enhance our understanding of gene regulatory networks, shedding new light on mammalian development, evolution and disease pathways. In this review, we provide a comprehensive overview of the intrinsic structural attributes, detection methodologies as well as the operational mechanisms of enhancers and promoters, coupled with the relevant novel and innovative investigative techniques used to explore their actions. We further elucidated the state-of-the-art research on the roles of enhancers and promoters in the realms of mammalian development, evolution and disease, and we conclude with forward-looking insights into prospective research avenues.

Programmed Cell Death in Unicellular Versus Multicellular Organisms.

Programmed cell death (self-induced) is intrinsic to all cellular life forms, including unicellular organisms. However, cell death research has focused on animal models to understand cancer, degenerative disorders, and developmental processes. Recently delineated suicidal death mechanisms in bacteria and fungi have revealed ancient origins of animal cell death that are intertwined with immune mechanisms, allaying earlier doubts that self-inflicted cell death pathways exist in microorganisms. Approximately 20 mammalian death pathways have been partially characterized over the last 35 years. By contrast, more than 100 death mechanisms have been identified in bacteria and a few fungi in recent years. However, cell death is nearly unstudied in most human pathogenic microbes that cause major public health burdens. Here, we consider how the current understanding of programmed cell death arose through animal studies and how recently uncovered microbial cell death mechanisms in fungi and bacteria resemble and differ from mechanisms of mammalian cell death.

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster.

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a model to study intestinal function, only a few reports have focused on Drosophila melanogaster (D. melanogaster). Compared to mice, fruit flies present many advantages, such as a short life cycle, cost-effective and simple maintenance, and no ethical issues. Furthermore, the mammalian gastrointestinal physiology, anatomy, and signaling pathways are highly conserved in D. melanogaster. Plant extracts have been used traditionally to treat diarrhea and constipation. For example, Psidium guajava (P. guajava) is one of the most known antidiarrheal agents in the tropics. However, no studies have evaluated the effect of antidiarrheal and laxative drugs and plant extracts in D. melanogaster, and it remains unknown if similar effects (e.g., smaller, more concentrated, and less abundant fecal deposits in the case of antidiarrheal drugs) can occur in the fruit flies compared to mammals. In this study, an antidiarrheal effect induced by P. guajava is demonstrated in a D. melanogaster strain that presents a diarrheic phenotype. Fecal sampling produced by flies is monitored using a dye-supplemented food. This protocol outlines the method used for preparing food with drugs, evaluating the fecal deposits of flies fed on these food preparations, and interpreting the data obtained.

Functional MRI activation of the nucleus tractus solitarius after taste stimuli at ultra-high field: a proof-of-concept single-subject study

Using ultra-high field (7 Tesla) functional MRI (fMRI), we conducted the first in-vivo functional neuroimaging study of the normal human brainstem specifically designed to examine neural signals in the Nucleus Tractus Solitarius (NTS) in response to all basic taste stimuli. NTS represents the first relay station along the mammalian taste processing pathway which originates at the taste buds in the oral cavity and passes through the thalamus before reaching the primary taste cortex in the brain. In our proof-of-concept study, we acquired data from one adult volunteer using fMRI at 1.2 mm isotropic resolution and performed a univariate general linear model analysis. During fMRI acquisition, three shuffled injections of sweet, bitter, salty, sour, and umami solutions were administered following an event-related design. We observed a statistically significant blood oxygen level-dependent (BOLD) response in the anatomically predicted location of the NTS for all five basic tastes. The results of this study appear statistically robust, even though they were obtained from a single volunteer. The information derived from a similar experimental strategy may inspire novel research aimed at clarifying important details of central nervous system involvement in eating disorders, at designing and monitoring tailored therapeutic strategies.

Stabilization of a Heme-HNO Model Complex Using a Bulky Bis-Picket Fence Porphyrin and Reactivity Studies with NO.

Nitroxyl, HNO/NO-, the one-electron reduced form of NO, is suggested to take part in distinct signaling pathways in mammals and is also a key intermediate in various heme-catalyzed NOx interconversions in the nitrogen cycle. Cytochrome P450nor (Cyt P450nor) is a heme-containing enzyme that performs NO reduction to N2O in fungal denitrification. The reactive intermediate in this enzyme, termed "Intermediate I", is proposed to be an Fe-NHO/Fe-NHOH type species, but it is difficult to study its electronic structure and exact protonation state due to its instability. Here, we utilize a bulky bis-picket fence porphyrin to obtain the first stable heme-HNO model complex, [Fe(3,5-Me-BAFP)(MI)(NHO)], as a model for Intermediate I, and more generally HNO adducts of heme proteins. Due to the steric hindrance of the bis-picket fence porphyrin, [Fe(3,5-Me-BAFP)(MI)(NHO)] is stable (τ1/2 = 56 min at -30 °C), can be isolated as a solid, and is available for thorough spectroscopic characterization. In particular, we were able to solve a conundrum in the literature and provide the first full vibrational characterization of a heme-HNO complex using IR and nuclear resonance vibrational spectroscopy (NRVS). Reactivity studies of [Fe(3,5-Me-BAFP)(MI)(NHO)] with NO gas show a 91 ± 10% yield for N2O formation, demonstrating that heme-HNO complexes are catalytically competent intermediates for NO reduction to N2O in Cyt P450nor. The implications of these results for the mechanism of Cyt P450nor are further discussed.

Identification of a New Drug Binding Site in the RNA-Dependent-RNA-Polymerase (RdRp) Domain

We hypothesize that in silico structural biology approaches can discover novel drug binding sites for RNA-dependent-RNA-polymerases (RdRp) of positive sense single-strand RNA (ss(+)RNA) virus species. RdRps have a structurally conserved active site with seven motifs (A to G), despite low sequence similarity. We refined this architecture further to describe a conserved structural domain consisting of motifs A, B, C and F. These motifs were used to realign 24 RdRp structures in an innovative manner to search for novel drug binding sites. The aligned motifs from the enzymes were then docked with 833 FDA-approved drugs (Set 1) and 85 FDA-approved antivirals (Set 2) using the Molecular Operating Environment (MOE) docking 2020.09 software. Sirolimus (rapamycin), an immunosuppressant that targets the mammalian mTOR pathway, was one of the top ten drugs for all 24 RdRp proteins. The sirolimus docking site was in the nucleotide triphosphate entry tunnel between motifs A and F but distinct from the active site in motif C. This original finding supports our hypothesis that structural biology approaches based on RdRp motifs that are conserved across evolution can define new drug binding locations and infer potential broad-spectrum inhibitors for SARS-CoV-2 and other ss(+)RNA viruses.

Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A.

A marine cyanobacterial cyclic depsipeptide, coibamide A (CbA), inhibits the mammalian protein secretory pathway by blocking the Sec61 translocon, which is an emerging drug target for cancer and other chronic diseases. In our previous structure-activity relationship study of CbA, the macrolactone ester linker was replaced with alkyl/alkenyl surrogates to provide synthetically accessible macrocyclic scaffolds. To optimize the cellular bioactivity profile of CbA analogues, novel lysine mimetics having β- and ε-methyl groups have now been designed and synthesized by a stereoselective route. A significant increase in cytotoxicity was observed upon introduction of these two methyl groups, corresponding to the d-MeAla α-methyl and MeThr β-methyl of CbA. All synthetic products retained the ability to inhibit secretion of a model Sec61 substrate. Tandem evaluation of secretory function inhibition in living cells and cytotoxicity was an effective strategy to assess the impact of structural modifications to the linker for ring closure.

Viral SERPINS-A Family of Highly Potent Immune-Modulating Therapeutic Proteins.

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2-10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics.

RNase L-activating 2'-5' oligoadenylates bind ABCF1, ABCF3 and Decr-1.

A notable signalling mechanism employed by mammalian innate immune signalling pathways uses nucleotide-based second messengers such as 2'3'-cGAMP and 2'-5'-oligoadenylates (OAs), which bind and activate STING and RNase L, respectively. Interestingly, the involvement of nucleotide second messengers to activate antiviral responses is evolutionarily conserved, as evidenced by the identification of an antiviral cGAMP-dependent pathway in Drosophila. Using a mass spectrometry approach, we identified several members of the ABCF family in human, mouse and Drosophila cell lysates as 2'-5' OA-binding proteins, suggesting an evolutionarily conserved function. Biochemical characterization of these interactions demonstrates high-affinity binding of 2'-5' OA to ABCF1, dependent on phosphorylated 2'-5' OA and an intact Walker A/B motif of the ABC cassette of ABCF1. As further support for species-specific interactions with 2'-5' OA, we additionally identified that the metabolic enzyme Decr1 from mouse, but not human or Drosophila cells, forms a high-affinity complex with 2'-5' OA. A 1.4 Å co-crystal structure of the mouse Decr1-2'-5' OA complex explains high-affinity recognition of 2'-5' OA and the mechanism of species specificity. Despite clear evidence of physical interactions, we could not identify profound antiviral functions of ABCF1, ABCF3 or Decr1 or 2'-5' OA-dependent regulation of cellular translation rates, as suggested by the engagement of ABCF proteins. Thus, although the biological consequences of the here identified interactions need to be further studied, our data suggest that 2'-5' OA can serve as a signalling hub to distribute a signal to different recipient proteins.

Relationship of tryptophan metabolites with the type and severity of multiple sclerosis.

Tryptophan is an essential amino acid primarily metabolized by the kynurenine pathway in mammals. Intermediate metabolites emerging in this pathway have been associated with many neurogenerative diseases. This study aimed to compare tryptophan pathway metabolite levels in patients with multiple sclerosis (MS) and healthy controls and reveal the relationship of tryptophan metabolites with disease subtype and the Expanded Disability Status Scale (EDSS) score. The study included a total of 80 MS cases [53 with relapsing remitting MS (RRMS) and 27 with secondary progressive MS (SPMS)] and 41 healthy volunteers. The patients with RRMS were further divided into relapse (RRMS-attack) and non-attack (RRMS-stable) groups. Using liquid chromatography mass spectrometry, tryptophan, kynurenine, kynurenic acid, quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid levels were measured. The serum metabolite levels of the patient and control groups were compared. In addition, the link and relationship between the EDSS score and disease duration of the patients and their plasma tryptophan metabolite levels were examined. The tryptophan level of the patient group was significantly lower than that of the healthy controls (p<0.05). The kynurenine (105.38±65.43), quinolinic acid (10.42±3.56), kynurenine/tryptophan ratio (0.0218±0.019), and quinolinic acid/kynurenic acid ratio (1.7054±0.96141) of the patients with MS were significantly higher compared to the controls (p<0.05). In the receiver operating characteristic analysis of the power of kynurenine/tryptophan and quinolinic acid/kynurenic acid ratios in predicting the disease, both ratios predicted the diagnosis of MS (area under the curve: 0.793 and 0.645, respectively; p<0.05), albeit at low sensitivity and specificity. The parameters were similar between the RRMS-attack and RRMS-stable patient groups (p>0.05). There was also no significant difference between the RRMS and SPMS patient groups in terms of tryptophan metabolites (p>0.05). Lastly, no significant relationship was observed between tryptophan metabolites and MS subtype and the EDSS score. Our findings revealed that the kynurenine pathway involved in the tryptophan metabolism differed between the patients with MS and healthy controls, and this difference may be a limited guide in the diagnosis of MS, due to major overlaps in values for MS versus Controls, and is insufficient to determine the disease subtype.

Circulating Sphingolipids and Glucose Homeostasis: An Update.

Sphingolipids are a family of lipid molecules produced through different pathways in mammals. Sphingolipids are structural components of membranes, but in response to obesity, they are implicated in the regulation of various cellular processes, including inflammation, apoptosis, cell proliferation, autophagy, and insulin resistance which favors dysregulation of glucose metabolism. Of all sphingolipids, two species, ceramides and sphingosine-1-phosphate (S1P), are also found abundantly secreted into the bloodstream and associated with lipoproteins or extracellular vesicles. Plasma concentrations of these sphingolipids can be altered upon metabolic disorders and could serve as predictive biomarkers of these diseases. Recent important advances suggest that circulating sphingolipids not only serve as biomarkers but could also serve as mediators in the dysregulation of glucose homeostasis. In this review, advances of molecular mechanisms involved in the regulation of ceramides and S1P association to lipoproteins or extracellular vesicles and how they could alter glucose metabolism are discussed.

The nuclei of the lateral lemniscus: unexpected players in the descending auditory pathway.

In the mammalian auditory pathway, the nuclei of the lateral lemniscus (NLL) are thought to be exclusively involved in the bottom-up transmission of auditory information. However, our repeated observation of numerous NLL neurons labeled after injection of retrograde tracers into the superior olivary complex (SOC) led us to systematically investigate with retrograde tracers the descending projections from the NLL to the SOC of the rat. We performed large injections of FluoroGold into the SOC to determine NLL contributions to descending projections, and focal injections of biotinylated dextran amine (BDA) to pinpoint the specific nuclei of the SOC innervated by each NLL. The SOC is innervated by thousands of neurons distributed across four nuclei or regions associated with the lateral lemniscus: the ipsilateral ventral and intermediate nuclei of the lateral lemniscus (VNLL and INLL); the medial paralemniscal region (PL) of both sides; and the ipsilateral semilunar nucleus (SLN), a previously unrecognized nucleus that wraps around the INLL dorsally, medially, and caudally and consists of small, flat neurons. In some experiments, at least 30% of neurons in the VNLL and INLL were retrogradely labeled. All nuclei of the SOC, except the medial and lateral superior olives, are innervated by abundant lemniscal neurons, and each SOC nucleus receives a unique combination of lemniscal inputs. The primary target of the projections from the VNLL is the ventral nucleus of the trapezoid body (VNTB), followed by the superior paraolivary nucleus (SPON), and the medial nucleus of the trapezoid body (MNTB). The INLL selectively innervates the VNTB. The PL innervates dorsal periolivary regions bilaterally. The SLN preferentially innervates the MNTB and may provide the first identified non-calyceal excitatory input to MNTB neurons. Our novel findings have strong implications for understanding acoustic information processing in the initial stages of the auditory pathway. Based on the proportion of lemniscal neurons involved in all the projections described, the NLL should be considered major players in the descending auditory pathway.

N-degron-mediated degradation of the proteolytically activated form of PKC-theta kinase attenuates its pro-apoptotic function

Cellular signalling cues lead to the initiation of apoptotic pathways and often result in the activation of caspases which in turn cause the generation of proteolytically generated protein fragments with new or altered functions. Mounting number of studies reveal that the activity of these proteolytically activated protein fragments can be counteracted via their selective degradation by the N-degron degradation pathways. Here, we investigate the proteolytically generated fragment of the PKC theta kinase, where we demonstrate the first report on the stability of this pro-apoptotic protein fragment. We have determined that the pro-apoptotic cleaved fragment of PKC-theta is unstable in cells because its N-terminal lysine targets it for proteasomal degradation via the N-degron degradation pathway and this degradation is inhibited by mutating the destabilizing N-termini, knockdown of the UBR1 and UBR2 E3 ligases. Tellingly, we demonstrate that the metabolic stabilization of the cleaved fragment of PKC-theta or inhibition of the N-degron degradation augments the apoptosis-inducing effect of staurosporine in Jurkat cells. Notably, we have unveiled that the cleaved fragment of PKC theta, per se, can induce apoptotic cell death in Jurkat T-cell leukemia. Our results expand the functional scope of mammalian N-degron degradation pathways, and support the notion that targeting N-degron degradation machinery may have promising therapeutic implications in cancer cells.

Identification of novel compounds against Acinetobacter baumannii 3-oxoacyl-[acyl-carrier-protein] synthase I (FabB) via comprehensive structure-based computational approaches

Acinetobacter baumannii is one of the most serious opportunistic pathogens according to WHO. The difference between bacterial and mammalian fatty acid biosynthesis pathways makes FASII enzymes attractive targets in drug discovery. 3-oxoacyl-[acyl-carrier-protein] synthase I (FabB) from the FAS II pathway catalyze the condensation of malonyl ACP with acyl-ACP, and elongates the fatty acid chain by two carbons. To investigate potential inhibitors of the A. baumannii FabB, we used computational approaches including homology modeling, high-throughput virtual screening, molecular docking, molecular dynamics simulations, and MM-GBSA free energy calculations. After the high-throughput virtual screening, the resulting ligands were further screened using the QM-polarized ligand docking (QPLD) and induced fit docking (IFD) approaches. Molecular dynamics simulations were performed for 100 ns. And according to binding free energy calculations, we have identified nine compounds with the best binding affinities. Three of these compounds were selected for an additional 1 μs MD simulation to assess ligand stability. Two of them named L6 and L7 showed promised stability and affinity to the target. Here, we present novel compounds against A. baumannii FabB via structure-based computational approaches. These compounds might pave the way for the design of new lead structures and inhibitors for multidrug-resistant A. baumannii.

Tale of cAMP as a second messenger in auxin signaling and beyond.

The 3',5'-cyclic adenosine monophosphate (cAMP) is a versatile second messenger in many mammalian signaling pathways. However, its role in plants remains not well-recognized. Recent discovery of adenylate cyclase (AC) activity for transport inhibitor response 1/auxin-signaling F-box proteins (TIR1/AFB) auxin receptors and the demonstration of its importance for canonical auxin signaling put plant cAMP research back into spotlight. This insight briefly summarizes the well-established cAMP signaling pathways in mammalian cells and describes the turbulent and controversial history of plant cAMP research highlighting the major progress and the unresolved points. We also briefly review the current paradigm of auxin signaling to provide a background for the discussion on the AC activity of TIR1/AFB auxin receptors and its potential role in transcriptional auxin signaling as well as impact of these discoveries on plant cAMP research in general.

Crosstalk between the mTOR and Hippo pathways.

Cell behavior changes in response to multiple stimuli, such as growth factors, nutrients, and cell density. The mechanistic target of rapamycin (mTOR) pathway is activated by growth factors and nutrient stimuli to regulate cell growth and autophagy, whereas the Hippo pathway has negative effects on cell proliferation and tissue growth in response to cell density, DNA damage, and hormonal signals. These two signaling pathways must be precisely regulated and integrated for proper cell behavior. This integrative mechanism is not completely understood; nevertheless, recent studies have suggested that components of the mTOR and Hippo pathways interact with each other. Herein, as per contemporary knowledge, we review the molecular mechanisms of the interaction between the mTOR and Hippo pathways in mammals and Drosophila. Moreover, we discuss the advantage of this interaction in terms of tissue growth and nutrient consumption. This article is protected by copyright. All rights reserved.

Phosphorylation of DNA-PKcs at the S2056 cluster ensures efficient and productive lymphocyte development in XLF-deficient mice

The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters-PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf -/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf -/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQRXlf -/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.

Design and Evolution of Enhanced Peptide-Peptide Ligation for Modular Transglutaminase Assembly.

Robust and precise tools are needed to enhance the functionality and resilience of synthetic nanoarchitectures. Here, we have employed directed evolution and rational design to build a fast-acting molecular superglue from a bacterial adhesion protein. We have generated the SnoopLigase2 coupling system, a genetically encoded route for efficient transamidation between SnoopTag2 and DogTag2 peptides. Each peptide was selected for rapid reaction by phage display screening. The optimized set allows more than 99% completion and is compatible with diverse buffers, pH values, and temperatures, accelerating the reaction over 1000-fold. SnoopLigase2 directs a specific reaction in the mammalian secretory pathway, allowing covalent display on the plasma membrane. Transglutaminase 2 (TG2) has a network of interactions and substrates amidst the mammalian cell surface and extracellular matrix. We expressed a modified TG2 with resistance to oxidative inactivation and minimal self-reactivity. SnoopLigase2 enables TG2 functionalization with transforming growth factor alpha (TGFα) in routes that would be impossible through genetic fusion. The TG2:TGFα conjugate retained transamidase activity, stably anchored TGFα for signal activation in the extracellular environment, and reprogrammed cell behavior. This modular toolbox should create new opportunities for molecular assembly, both for novel biomaterials and complex cellular environments.

The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3-8.3-fold and 19-22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10molecules that modulate the major mammalian DNA repair pathways identified twoinhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%-55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.