Microfluidic tool for rapid functional characterization of CRISPR complexes.

RNA guided nucleases are regarded as the future genome editing technologies. As such, they need to meet strong safety margins. Two major challenges in incorporating CRISPR technologies into the clinical world are off-target activity and editing efficiency. The common way to tackle such issues is to measure the binding and cleavage kinetics of the CRISPR enzyme. This can be challenging since, for example, DNA is not released from the CAS9 protein post cleavage. Here a promising new microfluidic approach to characterizing Enzymatic Interaction and Function of CRISPR complexes on a microfluidic platform (EnzyMIF) is presented. The method can rapidly detect the kd, koff, km and kcat for various RNA guided nucleases. In this work, two single guide RNAs with significantly different in-cell cleavage efficiency, RAG2 and RAG1, are used as proof-of-concept. The EnzyMIF assay results provide biochemical characterization of these guide RNAs that can explain the difference in cleavage using both wild type (WT) CAS9 and HiFi CAS9. Notably, it is shown that EnzyMIF characterization correlates with cell culture genomic editing efficiency results. It is suggested that EnzyMIF can predict the quality of cleavage rapidly and quantitatively.

Pseudouridines on Trypanosoma brucei mRNAs are developmentally regulated: Implications to mRNA stability and protein binding.

The parasite Trypanosoma brucei cycles between an insect and a mammalian host and is the causative agent of sleeping sickness. Here, we performed high-throughput mapping of pseudouridines (Ψs) on mRNA from two life stages of the parasite. The analysis revealed ~273 Ψs, including developmentally regulated Ψs that are guided by homologs of pseudouridine synthases (PUS1, 3, 5, and 7). Mutating the U that undergoes pseudouridylation in the 3' UTR of valyl-tRNA synthetase destabilized the mRNA level. To investigate the mechanism by which Ψ affects the stability of this mRNA, proteins that bind to the 3' UTR were identified, including the RNA binding protein RBSR1. The binding of RBSR1 protein to the 3' UTR was stronger when lacking Ψ compared to transcripts carrying the modification, suggesting that Ψ can inhibit the binding of proteins to their target and thus affect the stability of mRNAs. Consequently, Ψ modification on mRNA adds an additional level of regulation to the dominant post-transcriptional control in these parasites.

ß-blockers augment L-type Ca2+ channel activity by targeting spatially restricted ß2AR signaling in neurons.

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of ß-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate ß2AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated ß2AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant ß2AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant ß2AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which ß-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

Phosphodiesterase 5 Associates With ß2 Adrenergic Receptor to Modulate Cardiac Function in Type 2 Diabetic Hearts.

Background In murine heart failure models and in humans with diabetic-related heart hypertrophy, inhibition of phosphodiesterase 5 (PDE5) by sildenafil improves cardiac outcomes. However, the mechanism by which sildenafil improves cardiac function is unclear. We have observed a relationship between PDE5 and ß2 adrenergic receptor (ß2AR), which is characterized here as a novel mechanistic axis by which sildenafil improves symptoms of diabetic cardiomyopathy. Methods and Results Wild-type and ß2AR knockout mice fed a high fat diet (HFD) were treated with sildenafil, and echocardiogram analysis was performed. Cardiomyocytes were isolated for excitation-contraction (E-C) coupling, fluorescence resonant energy transfer, and proximity ligation assays; while heart tissues were implemented for biochemical and histological analyses. PDE5 selectively associates with ß2AR, but not ß1 adrenergic receptor, and inhibition of PDE5 with sildenafil restores the impaired response to adrenergic stimulation in HFD mice and isolated ventriculomyocytes. Sildenafil enhances ß adrenergic receptor (ßAR)-stimulated cGMP and cAMP signals in HFD myocytes. Consequently, inhibition of PDE5 leads to protein kinase G-, and to a lesser extent, calcium/calmodulin-dependent kinase II-dependent improvements in adrenergically stimulated E-C coupling. Deletion of ß2AR abolishes sildenafil's effect. Although the PDE5-ß2AR association is not altered in HFD, phosphodiesterase 3 displays an increased association with the ß2AR-PDE5 complex in HFD myocytes. Conclusions This study elucidates mechanisms by which the ß2AR-PDE5 axis can be targeted for treating diabetic cardiomyopathy. Inhibition of PDE5 enhances ß2AR stimulation of cGMP and cAMP signals, as well as protein kinase G-dependent E-C coupling in HFD myocytes.

Pseudouridines on Trypanosoma brucei spliceosomal small nuclear RNAs and their implication for RNA and protein interactions.

The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA-RNA and U2B"/U2A' proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics.

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.

Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.