Analysis of renal lesions in Chinese tuberous sclerosis complex patients with different types of TSC gene mutations

We sought to explore the relationship between renal lesion features and genetic mutations in tuberous sclerosis complex (TSC) patients. TSC patients with renal lesions were subjected to TSC1/2 gene next-generation sequencing (NGS). TSC1/2 mutation types and imaging examinations were screened for combined analysis of genetic and clinical features. Seventy-three probands among TSC patients with renal lesions were included. Twenty affected relatives were also included. In total, 93 patients were included. Eighty patients (86.0%) had bilateral renal angiomyolipomas (AMLs), and one had epithelioid AML. Two patients had polycystic kidney disease, one had renal cell carcinoma, and one had Wilms tumor. Among the 73 probands, four had TSC1 mutations, 53 had TSC2 mutations, and 16 had no mutations identified (NMI). There was no statistically significant difference between TSC1 mutation, TSC2 mutation and NMI group (P= 0.309), or between familial and sporadic groups (P= 0.775) when considering AML size. There was no statistically significant difference between pathogenic/likely pathogenic and benign/likely benign/NMI groups (P= 0.363) or among patients with different mutation types of TSC2 (P= 0.906). The relationship between the conditions of TSC gene mutations and the severity of renal lesions still needs more analysis. Patients with NMI, particularly those with familial disease, need more attention because the pathogenesis remains unknown.     

Phosphorylation and binding partner analysis of the TSC1-TSC2 complex

Tuberous sclerosis complex (TSC) is an autosomal dominant benign tumour syndrome caused by mutations to either the TSC1 or TSC2 tumour suppressor gene. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a protein complex that integrates inputs from multiple signalling cascades to inactivate the small GTPase rheb, and thereby inhibit mTOR-dependent cell growth. We have used matrix-assisted laser desorption/ionisation time-of-flight and Fourier transform mass spectrometry to identify TSC1 and TSC2 phosphorylation sites and candidate TSC1 and TSC2 interacting proteins. We identified three sites of TSC2 phosphorylation and a novel site of TSC1 phosphorylation, and investigated the roles of these sites in regulating the activity of the TSC1-TSC2 complex. In addition, we identified three TSC1-TSC2 interacting proteins, including DOCK7 a putative rhebGEF.     

Tuberous sclerosis complex 2 loss-of-function mutation regulates reactive oxygen species production through Rac1 activation

The products of the TSC1 (hamartin) and TCS2 (tuberin) tumor suppressor genes negatively regulate cell growth by inhibiting mTOR signaling. Recent research has led to the postulation that tuberin and/or hamartin are involved in tumor migration, presumably through Rho activation. Here we show that LEF-8 cells, which contain a Y1571 missense mutation in tuberin, express higher Rac1 activity than tuberin negative and positive cells. We also provide evidence of obvious lamellipodia formation in LEF-8 cells. Since the production of TSC2^Y1571H cannot form a hetero-complex with hamartin, we further analyzed another mutant, TSC2^R611Q, which also lacks the ability to form a complex with hamartin. Introducing both forms of mutated TSC2 into COS-1 cells increased Rac1 activity as well as cell motility. We also found these two mutants interacted with Rac1. We further demonstrated that the introduction of mutated TSC2 into COS-1 cells can generate higher reactive oxygen species (ROS). These results indicate that loss-of-function mutated tuberin can activate Rac1 and thereby increase ROS production.     

Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.     

Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency

VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.     

CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution

Despite previous developmental studies on basally branching wingless insects and crustaceans, the evolutionary origin of insect wings remains controversial. Knowledge regarding genetic regulation of tissues hypothesized to have given rise to wings would help to elucidate how ancestral development changed to allow the evolution of true wings. However, genetic tools available for basally branching wingless species are limited. The firebrat Thermobia domestica is an apterygote species, phylogenetically related to winged insects. T. domestica presents a suitable morphology to investigate the origin of wings, as it forms the tergal paranotum, from which wings are hypothesized to have originated. Here we report the first successful CRISPR/Cas9-based germline genome editing in T. domestica. We provide a technological platform to understand the development of tissues hypothesized to have given rise to wings in an insect with a pre-wing evolution body plan.     

Developing CRISPR-based sex-ratio distorters for the genetic control of fruit fly pests: A how to manual

Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes.     

Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins

Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops. Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action. Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence‐based gene silencing approaches. In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H. armigera to establish three homozygous knockout strains. Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera.     

Axin2‐mTurquoise2: A novel reporter mouse model for the detection of canonical Wnt signalling

The canonical Wnt signalling pathway has been implicated in organogenesis and self‐renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR‐Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli‐β‐galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2‐TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.     

Fluorescent indicators for continuous and lineage-specific reporting of cell-cycle phases in human pluripotent stem cells

Proper cell-cycle progression is essential for the self-renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination-based cell-cycle indicator (FUCCI) has allowed the dual-color visualization of the G₁ and S/G₂/M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage-specific FUCCI reporters have not yet been developed to analyze complex tissue-specific cell-cycle progression during hPSC differentiation. Desiring a robust tool for spatiotemporal reporting of cell-cycle events in hPSCs, we employed the CRISPR/Cas9 genome editing tool and successfully knocked the FUCCI reporter into the AAVS1 safe harbor locus of hPSCs for stable and constitutive FUCCI expression, exhibiting reliable cell-cycle-dependent fluorescence in both hPSCs and their differentiated progeny. We also established a cardiac-specific TNNT2-FUCCI reporter for lineage-specific cell-cycle monitoring of cardiomyocyte differentiation from hPSCs. This powerful and modular FUCCI system should provide numerous opportunities for studying human cell-cycle activity, and enable the identification and investigation of novel regulators for adult tissue regeneration.     

CRISPR/Cas9: A new tool for the study and control of helminth parasites

Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9‐targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9‐mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.     

Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1

CRISPR‐Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non‐histone chromosomal protein HMG‐14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA‐MS) is utilized, and more than 6200 proteins (protein‐ FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA‐MS in all of the clone‐ and dish‐ replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA‐MS can be reliably used as a rapid, robust, and cost‐effective proteomic‐screening tool to assess the outcome of the CRISPR experiments.     

Recombinase-mediated cassette exchange-based screening of a CRISPR/Cas9 library for enhanced recombinant protein production in human embryonic kidney cells: Improving resistance to hyperosmotic stress

Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.