GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells.

Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome-wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to 5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.

Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA.

CRISPR-based gene editing in mammalian cells is a powerful research tool which has demonstrated efficient site-specific gene modifications and is showing promise as a therapeutic for patients with genetic diseases. Multiple different CRISPR systems have been identified, each with its own target DNA recognition sequence, expanding the editable mammalian genome. It is also now appreciated that chemically modified nucleic acids can substitute for unmodified nucleotides in guide RNAs, providing protection from exonuclease degradation and improving gene editing efficiency. CRISPR-Cpf1 unlike CRISPR-Cas9, has a substantially lower propensity for off-target genomic cleavage, making it a preferred gene editing system for many applications. Here we provide a detailed protocol for use of CRISPR-Cpf1 and chemically modified guide RNAs in cell lines, outlining the steps from designing guide RNAs to a target gene of interest, delivery and expression in cells, and analysis of gene editing events.

Kinetic and subcellular analysis of PS-ASO/protein interactions with P54nrb and RNase H1.

The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein-protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO-protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.

Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.

The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2'-O-methoxyethyl (2'-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1-dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2'-O-methyl (2'-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles.

Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells.

CRISPR-based gene editing is a powerful technology for engineering mammalian genomes. It holds the potential as a therapeutic, although much-needed in vivo delivery systems have yet to be established. Here, using the Cpf1-crRNA (CRISPR RNA) crystal structure as a guide, we synthesized a series of systematically truncated and chemically modified crRNAs, and identify positions that are amenable to modification while retaining gene-editing activity. Modified crRNAs were designed with the same modifications that provide protection against nucleases and enable wide distribution in vivo. We show crRNAs with chemically modified terminal nucleotides are exonuclease resistant while retaining gene-editing activity. Chemically modified or DNA-substituted nucleotides at select positions and up to 70% of the crRNA DNA specificity region are also well tolerated. In addition, gene-editing activity is maintained with phosphorothioate backbone substitutions in the crRNA DNA specificity region. Finally, we demonstrate that 42-mer synthetic crRNAs from the similar CRISPR-Cas9 system are taken up by cells, an attractive property for in vivo delivery. Our study is the first to show that chemically modified crRNAs of the CRISPR-Cpf1 system can functionally replace and mediate comparable gene editing to the natural crRNA, which holds the potential for enhancing both viral- and non-viral-mediated in vivo gene editing.

TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

A secretory kinase complex regulates extracellular protein phosphorylation.

Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation.

Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.

Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2-3Galß1-4Xylß1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.

Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis.

The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380-385) and these phosphorylations are proposed to induce a reduction in PTEN's plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN's regulation and suggest pharmacologic approaches for direct PTEN activation. DOI:http://dx.doi.org/10.7554/eLife.00691.001.

A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN.

The PI 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), one of the most important tumor suppressors, must associate with the plasma membrane to maintain appropriate steady-state levels of phosphatidylinositol 3,4,5-triphosphate. Yet the mechanism of membrane binding has received little attention and the key determinants that regulate localization, a phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding motif and a cluster of phosphorylated C-terminal residues, were not included in the crystal structure. We report that membrane binding requires PIP(2) and show that phosphorylation regulates an intramolecular interaction. A truncated version of the enzyme, PTEN(1-351), bound strongly to the membrane, an effect that was reversed by co-expression of the remainder of the molecule, PTEN(352-403). The separate fragments associated in vitro, an interaction dependent on phosphorylation of the C-terminal cluster, a portion of the PIP(2) binding motif, integrity of the phosphatase domain, and the CBR3 loop. Our investigation provides direct evidence for a model in which PTEN switches between open and closed states and phosphorylation favors the closed conformation, thereby regulating localization and function. Small molecules targeting these interactions could potentially serve as therapeutic agents in antagonizing Ras or PI3K-driven tumors. The study also stresses the importance of determining the structure of the native enzyme.

Live-cell molecular analysis of Akt activation reveals roles for activation loop phosphorylation.

Activation of the serine/threonine protein kinase Akt/PKB is a multi-step process involving membrane recruitment, phosphorylation, and membrane detachment. To investigate this process in the cellular context, we employed a live-cell fluorescence imaging approach to examine conformational changes of Akt and its membrane association. A fluorescence resonance energy transfer-based reporter of Akt action (ReAktion) reveals a conformational change that is critically dependent on the existence of a phosphorylatable threonine 308 in the activation loop, because mutations to either aspartate or alanine abolished the change. Furthermore, a mutant carrying a phosphorylation mimic at this position showed diminished membrane association, suggesting that this phosphorylation plays an important role of promoting the dissociation of activated Akt from the membrane. In addition, the membrane-associating pleckstrin homology domain was found to associate with the catalytic domain when Thr308 is phosphorylated, suggesting such an interdomain interaction as a mechanism by which phosphorylation within the catalytic domain can affect membrane association. These studies uncover new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.

Reading dynamic kinase activity in living cells for high-throughput screening.

Protein kinases, as crucial signaling molecules, represent an emerging class of drug targets, and the ability to assay their activities in living cells with high-throughput screening should provide exciting opportunities for drug discovery and chemical and functional genomics. Here, we describe a general method for high-throughput reading of dynamic kinase activities using ratiometric fluorescent sensors, and showcase an example of reading intracellular activities of protein kinase A (PKA) and the cyclic adenosine monophosphate (cAMP)/PKA pathway downstream of many G-protein coupled receptors (GPCRs). We further demonstrate the first compound screen based on the ability of compounds to modulate dynamic kinase activities in living cells and show that such screening of a collection of clinical compounds has successfully identified modulators of the GPCR/cAMP/PKA pathway.