Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays.

Genomewide loss-of-function (LOF) screening using clustered regularly interspaced short palindromic repeats (CRISPR) has facilitated the discovery of novel gene functions across diverse physiological and pathophysiological systems. A challenge with conventional genomewide CRISPR-Cas9 libraries is the unwieldy size (60,000-120,000 constructs), which is resource intensive and prohibitive in some experimental contexts. One solution to streamlining CRISPR screening is by multiplexing two or more guides per gene on a single construct, which enables functional redundancy to compensate for suboptimal gene knockout by individual guides. In this regard, AsCas12a (Cpf1) and its derivatives, for example, enhanced AsCas12a (enAsCas12a), have enabled multiplexed guide arrays to be specifically and efficiently processed for genome editing. Prior studies have established that multiplexed CRISPR-Cas12a libraries perform comparably to the larger equivalent CRISPR-Cas9 libraries, yet the most efficient CRISPR-Cas12a library design remains unresolved. In this study, we demonstrate that CRISPR-Cas12a genomewide LOF screening performed optimally with three guides arrayed per gene construct and could be adapted to robotic cell culture without noticeable differences in screen performance. Thus, the conclusions from this study provide novel insight to streamlining genomewide LOF screening using CRISPR-Cas12a and robotic cell culture.

Computation Offloading Game for Multi-Channel Wireless Sensor Networks.

Computation offloading for wireless sensor devices is critical to improve energy efficiency and maintain service delay requirements. However, simultaneous offloadings may cause high interferences to decrease the upload rate and cause additional transmission delay. It is thus intuitive to distribute wireless sensor devices in different channels, but the problem of multi-channel computation offloading is NP-hard. In order to solve this problem efficiently, we formulate the computation offloading decision problem as a decision-making game. Then, we apply the game theory to address the problem of allowing wireless sensor devices to make offloading decisions based on their own interests. In the game theory, not only are the data size of wireless sensor devices and their computation capability considered but the channel gain of each wireless sensor device is also included to improve the transmission rate. The consideration could evenly distribute wireless sensor devices to different channels. We prove that the proposed offloading game is a potential game, where the Nash equilibrium exists in each game after all device states converge. Finally, we extensively evaluate the performance of the proposed algorithm based on simulations. The simulation results demonstrate that our algorithm can reduce the number of iterations to achieve Nash equilibrium by 16%. Moreover, it improves the utilization of each channel to effectively increase the number of successful offloadings and lower the energy consumption of wireless sensor devices.

Trajectories of Homoeolog-Specific Expression in Allotetraploid Tragopogon castellanus Populations of Independent Origins.

Polyploidization can have a significant ecological and evolutionary impact by providing substantially more genetic material that may result in novel phenotypes upon which selection may act. While the effects of polyploidization are broadly reviewed across the plant tree of life, the reproducibility of these effects within naturally occurring, independently formed polyploids is poorly characterized. The flowering plant genus Tragopogon (Asteraceae) offers a rare glimpse into the intricacies of repeated allopolyploid formation with both nascent (< 90 years old) and more ancient (mesopolyploids) formations. Neo- and mesopolyploids in Tragopogon have formed repeatedly and have extant diploid progenitors that facilitate the comparison of genome evolution after polyploidization across a broad span of evolutionary time. Here, we examine four independently formed lineages of the mesopolyploid Tragopogon castellanus for homoeolog expression changes and fractionation after polyploidization. We show that expression changes are remarkably similar among these independently formed polyploid populations with large convergence among expressed loci, moderate convergence among loci lost, and stochastic silencing. We further compare and contrast these results for T. castellanus with two nascent Tragopogon allopolyploids. While homoeolog expression bias was balanced in both nascent polyploids and T. castellanus, the degree of additive expression was significantly different, with the mesopolyploid populations demonstrating more non-additive expression. We suggest that gene dosage and expression noise minimization may play a prominent role in regulating gene expression patterns immediately after allopolyploidization as well as deeper into time, and these patterns are conserved across independent polyploid lineages.

Linked read technology for assembling large complex and polyploid genomes.

BACKGROUND: Short read DNA sequencing technologies have revolutionized genome assembly by providing high accuracy and throughput data at low cost. But it remains challenging to assemble short read data, particularly for large, complex and polyploid genomes. The linked read strategy has the potential to enhance the value of short reads for genome assembly because all reads originating from a single long molecule of DNA share a common barcode. However, the majority of studies to date that have employed linked reads were focused on human haplotype phasing and genome assembly. RESULTS: Here we describe a de novo maize B73 genome assembly generated via linked read technology which contains ~ 172,000 scaffolds with an N50 of 89 kb that cover 50% of the genome. Based on comparisons to the B73 reference genome, 91% of linked read contigs are accurately assembled. Because it was possible to identify errors with > 76% accuracy using machine learning, it may be possible to identify and potentially correct systematic errors. Complex polyploids represent one of the last grand challenges in genome assembly. Linked read technology was able to successfully resolve the two subgenomes of the recent allopolyploid, proso millet (Panicum miliaceum). Our assembly covers ~ 83% of the 1 Gb genome and consists of 30,819 scaffolds with an N50 of 912 kb. CONCLUSIONS: Our analysis provides a framework for future de novo genome assemblies using linked reads, and we suggest computational strategies that if implemented have the potential to further improve linked read assemblies, particularly for repetitive genomes.

L-Met Activates Arabidopsis GLR Ca2+ Channels Upstream of ROS Production and Regulates Stomatal Movement.

Plant glutamate receptor homologs (GLRs) have long been proposed to function as ligand-gated Ca2+ channels, but no in planta evidence has been provided. Here, we present genetic evidence that Arabidopsis GLR3.1 and GLR3.5 form Ca2+ channels activated by L-methionine (L-Met) at physiological concentrations and regulate stomatal apertures and plant growth. The glr3.1/3.5 mutations resulted in a lower cytosolic Ca2+ level, defective Ca2+-induced stomatal closure, and Ca2+-deficient growth disorder, all of which involved L-Met. Patch-clamp analyses of guard cells showed that GLR3.1/3.5 Ca2+ channels are activated specifically by L-Met, with the activation abolished in glr3.1/3.5. Moreover, GLR3.1/3.5 Ca2+ channels are distinct from previously characterized ROS-activated Ca2+ channels and act upstream of ROS, providing Ca2+ transients necessary for the activation of NADPH oxidases. Our data indicate that GLR3.1/3.5 constitute L-Met-activated Ca2+ channels responsible for maintaining basal [Ca2+]cyt, play a pivotal role in plant growth, and act upstream of ROS, thereby regulating stomatal aperture.

Characterization of maize roothairless6 which encodes a D-type cellulose synthase and controls the switch from bulge formation to tip growth.

Root hairs are tubular extensions of the epidermis. Root hairs of the monogenic recessive maize mutant roothairless 6 (rth6) are arrested after bulge formation during the transition to tip growth and display a rough cell surface. BSR-Seq in combination with Seq-walking and subsequent analyses of four independently generated mutant alleles established that rth6 encodes CSLD5 a plasma membrane localized 129 kD D-type cellulose synthase with eight transmembrane domains. Cellulose synthases are required for the biosynthesis of cellulose, the most abundant biopolymer of plant cell walls. Phylogenetic analyses revealed that RTH6 is part of a monocot specific clade of D-type cellulose synthases. D-type cellulose synthases are highly conserved in the plant kingdom with five gene family members in maize and homologs even among early land plants such as the moss Physcomitrella patens or the clubmoss Selaginella moellendorffii. Expression profiling demonstrated that rth6 transcripts are highly enriched in root hairs as compared to all other root tissues. Moreover, in addition to the strong knock down of rth6 expression in young primary roots of the mutant rth6, the gene is also significantly down-regulated in rth3 and rth5 mutants, while it is up-regulated in rth2 mutants, suggesting that these genes interact in cell wall biosynthesis.

Genetic control of morphometric diversity in the maize shoot apical meristem.

The maize shoot apical meristem (SAM) comprises a small pool of stem cells that generate all above-ground organs. Although mutational studies have identified genetic networks regulating SAM function, little is known about SAM morphological variation in natural populations. Here we report the use of high-throughput image processing to capture rich SAM size variation within a diverse maize inbred panel. We demonstrate correlations between seedling SAM size and agronomically important adult traits such as flowering time, stem size and leaf node number. Combining SAM phenotypes with 1.2 million single nucleotide polymorphisms (SNPs) via genome-wide association study reveals unexpected SAM morphology candidate genes. Analyses of candidate genes implicated in hormone transport, cell division and cell size confirm correlations between SAM morphology and trait-associated SNP alleles. Our data illustrate that the microscopic seedling SAM is predictive of adult phenotypes and that SAM morphometric variation is associated with genes not previously predicted to regulate SAM size.

Border control--a membrane-linked interactome of Arabidopsis.

Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 10(6) pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split-green flourescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

The maize glossy13 gene, cloned via BSR-Seq and Seq-walking encodes a putative ABC transporter required for the normal accumulation of epicuticular waxes.

Aerial plant surfaces are covered by epicuticular waxes that among other purposes serve to control water loss. Maize glossy mutants originally identified by their "glossy" phenotypes exhibit alterations in the accumulation of epicuticular waxes. By combining data from a BSR-Seq experiment and the newly developed Seq-Walking technology, GRMZM2G118243 was identified as a strong candidate for being the glossy13 gene. The finding that multiple EMS-induced alleles contain premature stop codons in GRMZM2G118243, and the one knockout allele of gl13, validates the hypothesis that gene GRMZM2G118243 is gl13. Consistent with this, GRMZM2G118243 is an ortholog of AtABCG32 (Arabidopsis thaliana), HvABCG31 (barley) and OsABCG31 (rice), which encode ABCG subfamily transporters involved in the trans-membrane transport of various secondary metabolites. We therefore hypothesize that gl13 is involved in the transport of epicuticular waxes onto the surfaces of seedling leaves.

Calcium-permeable channels in plant cells.

Calcium signal transduction is a central mechanism by which plants sense and respond to endogenous and environmental stimuli. Cytosolic Ca(2+) elevation is achieved via two cellular pathways, Ca(2+) influx through Ca(2+) channels in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Because of the significance of Ca(2+) channels in cellular signaling, interaction with the environment and developmental processes in plants, a great deal of effort has been invested in recent years with regard to these important membrane proteins. Because of limited space, in this review we focus on recent findings giving insight into both the molecular identity and physiological function of channels that have been suggested to be responsible for the elevation in cytosolic Ca(2+) level, including cyclic nucleotide gated channels, glutamate receptor homologs, two-pore channels and mechanosensitive Ca(2+) -permeable channels. We provide an overview of the regulation of these Ca(2+) channels and their physiological roles and discuss remaining questions.

Arabidopsis annexins AnnAt1 and AnnAt4 interact with each other and regulate drought and salt stress responses.

Annexins are Ca2+--and phospholipid-binding proteins that form an evolutionarily conserved multigene family throughout the animal and plant kingdoms. Two annexins, AnnAt1 and AnnAt4, have been identified as components in osmotic stress and abscisic acid signaling in Arabidopsis. Here, we report that AnnAt1 and AnnAt4 regulate plant stress responses in a light-dependent manner. The single-mutant annAt1 and annAt4 plants showed tolerance to drought and salt stress, which was greatly enhanced in double-mutant annAt1annAt4 plants, but AnnAt4-overexpressing transgenic plants (35S:AnnAt4) were more sensitive to stress treatments under long day conditions. Furthermore, expression of stress-related genes was altered in these mutant and transgenic plants. Upon dehydration and salt treatment, AtNCED3, encoding 9-cis-epoxycarotenoid dioxygenase, and P5CS1, encoding Δ-1-pyrroline-5-carboxylate synthase, which are key enzymes in ABA and proline synthesis, respectively, were highly induced in annAt1annAt4 plants and to a lesser extent in annAt1 and annAt4 plants, but not in 35S:AnnAt4 plants. While annAt1 plants were more drought sensitive, annAt4 plants were more tolerant in short days than in long days. In vitro and in vivo binding assays revealed that AnnAt1 and AnnAt4 bind to each other in a Ca2+-dependent manner. Our results suggest that AnnAt1 and AnnAt4 function cooperatively in response to drought and salt stress and their functions are affected by photoperiod.

Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase.

The plant hormone abscisic acid (ABA) triggers production of reactive oxygen species (ROS) in guard cells via the AtrbohD and AtrbohF NADPH oxidases, leading to stomatal closure. The ABA-activated SnRK2 protein kinase open stomata 1 (OST1) (SRK2E/SnRK2.6) acts upstream of ROS in guard cell ABA signaling. Here, we report that OST1 phosphorylates Ser13 and Ser174 on AtrbohF. In addition, substitution of Ser174 to Ala results in a approximately 40% reduction in the phosphorylation of AtrbohF by OST1. We also show that OST1 physically interacts with AtrbohF. These results provide biochemical evidence suggesting that OST1 regulates AtrbohF activity.

CHL1 functions as a nitrate sensor in plants.

Ions serve as essential nutrients in higher plants and can also act as signaling molecules. Little is known about how plants sense changes in soil nutrient concentrations. Previous studies showed that T101-phosphorylated CHL1 is a high-affinity nitrate transporter, whereas T101-dephosphorylated CHL1 is a low-affinity transporter. In this study, analysis of an uptake- and sensing-decoupled mutant showed that the nitrate transporter CHL1 functions as a nitrate sensor. Primary nitrate responses in CHL1T101D and CHLT101A transgenic plants showed that phosphorylated and dephosphorylated CHL1 lead to a low- and high-level response, respectively. In vitro and in vivo studies showed that, in response to low nitrate concentrations, protein kinase CIPK23 can phosphorylate T101 of CHL1 to maintain a low-level primary response. Thus, CHL1 uses dual-affinity binding and a phosphorylation switch to sense a wide range of nitrate concentrations in the soil, thereby functioning as an ion sensor in higher plants. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

AtCIPK8, a CBL-interacting protein kinase, regulates the low-affinity phase of the primary nitrate response.

Nitrate, the major nitrogen source for most plants, is not only a nutrient but also a signaling molecule. For almost two decades, it has been known that nitrate can rapidly induce transcriptional expression of several nitrate-related genes, a process that is referred to as the primary nitrate response. However, little is known about how plants actually sense nitrate and how the signal is transmitted in this pathway. In this study, a calcineurin B-like (CBL) -interacting protein kinase (CIPK) gene, CIPK8, was found to be involved in early nitrate signaling. CIPK8 expression was rapidly induced by nitrate. Analysis of two independent knockout mutants and a complemented line showed that CIPK8 positively regulates the nitrate-induced expression of primary nitrate response genes, including nitrate transporter genes and genes required for assimilation. Kinetic analysis of nitrate induction levels of these genes in wild-type plants indicated that there are two response phases: a high-affinity phase with a K(m) of approximately 30 mum and a low-affinity phase with a K(m) of approximately 0.9 mm. As cipk8 mutants were defective mainly in the low-affinity response, the high-affinity and low-affinity nitrate signaling systems are proposed to be genetically distinct, with CIPK8 involved in the low-affinity system. In addition, CIPK8 was found to be involved in long-term nitrate-modulated primary root growth and nitrate-modulated expression of a vacuolar malate transporter. Taken together, our results indicate that CBL-CIPK networks are responsible not only for stress responses and potassium shortage, but also for nitrate sensing.

Genetic toxicity of methamphetamine in vitro and in human abusers.

Methamphetamine (METH) is a widely abused psychomotor stimulant. Although numerous studies have examined METH-induced neurotoxicity, its ability to produce genotoxic effects has not been evaluated. In this article, we report on the genotoxicity of METH in vitro and in human METH abusers. METH induced his(+) revertants in Salmonella typhimurium strains TA98 and TA100, and increased the frequency of hprt mutants, micronuclei, and sister chromatid exchange (SCE) in cultured Chinese hamster ovary K1 (CHO-K1) cells. These METH-induced genotoxic effects were eliminated if METH exposure was conducted in the presence of rat liver S9, indicating that the genotoxicity was caused by METH, and not by metabolites of METH. In addition, reactive oxygen species (ROS) scavengers inhibited the METH-induced micronuclei in CHO-K1 cells. Further investigation with 76 human long-term METH abusers and 98 unexposed controls demonstrated that total METH exposure correlated with micronucleus and SCE frequencies in cultured lymphocytes. The results of this study indicate that METH is a genotoxic agent and that ROS may play a role in METH-induced genotoxicity.