Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments.

The CRISPR/Cas9 system has enabled efficient modification of genes in a variety of cellular systems for studying phenotypic effects of genetic perturbations. However, with this technology comes the inherent risk of generating off-target effects (OTEs) in addition to the desired modifications. As such, it can be difficult to conclusively determine that the observed phenotypic changes are in fact due to the intended modification of the target gene and not from random mutations elsewhere in the genome. In addition, biological variations observed within cultured cells or laboratory animals can also confound results and need to be addressed. In this article, we review potential sources of experimental and biological variation as well as propose experimental options to minimize and control OTEs and other variations in CRISPR genome editing experiments for exploratory research applications. Confirmation of on-target KO effect by orthogonal approaches is also discussed.

T Cells Encountering Myeloid Cells Programmed for Amino Acid-dependent Immunosuppression Use Rictor/mTORC2 Protein for Proliferative Checkpoint Decisions.

Modulation of T cell proliferation and function by immunoregulatory myeloid cells are an essential means of preventing self-reactivity and restoring tissue homeostasis. Consumption of amino acids such as arginine and tryptophan by immunoregulatory macrophages is one pathway that suppresses local T cell proliferation. Using a reduced complexity in vitro macrophage-T cell co-culture system, we show that macrophage arginase-1 is the only factor required by M2 macrophages to block T cells in G1, and this effect is mediated by l-arginine elimination rather than metabolite generation. Tracking how T cells adjust their metabolism when deprived of arginine revealed the significance of macrophage-mediated arginine deprivation to T cells. We found mTORC1 activity was unaffected in the initial G1 block. After 2 days of arginine deprivation, mTORC1 activity declined paralleling a selective down-regulation of SREBP target gene expression, whereas mRNAs involved in glycolysis, gluconeogenesis, and T cell activation were unaffected. Cell cycle arrest was reversible at any point by exogenous arginine, suggesting starved T cells remain poised awaiting nutrients. Arginine deprivation-induced cell cycle arrest was mediated in part by Rictor/mTORC2, providing evidence that this nutrient recognition pathway is a central component of how T cells measure environmental arginine.

Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma.

Transdifferentiation (TD) is a recent advancement in somatic cell reprogramming. The direct conversion of TD eliminates the pluripotent intermediate state to create cells that are ideal for personalized cell therapy. Here we provide evidence that TD-derived induced neural stem cells (iNSCs) are an efficacious therapeutic strategy for brain cancer. We find that iNSCs genetically engineered with optical reporters and tumouricidal gene products retain the capacity to differentiate and induced apoptosis in co-cultured human glioblastoma cells. Time-lapse imaging shows that iNSCs are tumouritropic, homing rapidly to co-cultured glioblastoma cells and migrating extensively to distant tumour foci in the murine brain. Multimodality imaging reveals that iNSC delivery of the anticancer molecule TRAIL decreases the growth of established solid and diffuse patient-derived orthotopic glioblastoma xenografts 230- and 20-fold, respectively, while significantly prolonging the median mouse survival. These findings establish a strategy for creating autologous cell-based therapies to treat patients with aggressive forms of brain cancer.

Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice.

BACKGROUND: Pantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in PANK2, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in PANK2 reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease. OBJECTIVE, METHODS, RESULTS AND CONCLUSIONS: Cellular CoA levels are tightly regulated by a balance between synthesis and degradation. CoA degradation is catalyzed by two peroxisomal nudix hydrolases, Nudt7 and Nudt19. In this study we sought to reduce neuronal CoA in mice through the alternative approach of increasing Nudt7-mediated CoA degradation. This was achieved by combining the use of an adeno-associated virus-based expression system with the synapsin (Syn) promoter. We show that mice with neuronal overexpression of a cytosolic version of Nudt7 (scAAV9-Syn-Nudt7cyt) exhibit a significant decrease in brain CoA levels in conjunction with a reduction in motor coordination. These results strongly support the existence of a link between CoA levels and neuronal function and show that scAAV9-Syn-Nudt7cyt mice can be used to model PKAN.

Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein.

The angiotensin II type 1 receptor (AT(1)R) mediates most hypertensive actions of angiotensin II. To understand the molecular regulation of the AT(1)R in normal physiology and pathophysiology, methods for sensitive and specific detection of AT(1)R protein are required. Here, we examined the specificity of a panel of putative AT(1)R antibodies that are commonly used by investigators in the field. For these studies, we carried out Western blotting and immunohistochemistry with kidney tissue from wild-type mice and genetically modified mice lacking the major murine AT(1)R isoform, AT(1A) (AT(1A)KO), or with combined deficiency of both the AT(1A) and AT(1B) isoforms (AT(1AB)KO). For the 3 antibodies tested, Western blots of protein homogenates from wild-type kidneys yielded distinct bands with the expected size range for AT(1)R. In addition, these bands appeared identical in samples from mice lacking 1 or both murine AT(1)R isoforms. Additionally, the pattern of immunohistochemical staining in kidneys, liver, and adrenal glands of wild-type mice was very similar to that of AT(1AB)KO mice completely lacking all AT(1)R. We verified the absence of AT(1)R subtypes in each mouse line by the following: (1) quantitative polymerase chain reaction documenting the absence of mRNA species, and (2) functionally by assessing angiotensin II-dependent vasoconstriction, which was substantially blunted in both AT(1A)KOs and AT(1AB)KOs. Finally, these antibodies failed to detect epitope-tagged AT(1A)R protein overexpressed in human embryonic kidney cells. We conclude that anti-AT(1)R antibodies available from commercial sources and commonly used in published studies exhibit nonspecific binding in mouse tissue that may lead to erroneous results.

Compartmentalization of mammalian pantothenate kinases.

The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1ß and PanK3 isoforms were cytosolic, with a portion of PanK1ß associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.

c-Fos activates and physically interacts with specific enzymes of the pathway of synthesis of polyphosphoinositides.

The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II ß was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.

c-Fos activated phospholipid synthesis is required for neurite elongation in differentiating PC12 cells.

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.