Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA.

Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR-Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3-5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.

Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs.

CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.

Neural Net Classification Combined With Movement Analysis to Evaluate Setaria viridis as a Model System for Time of Day of Anther Appearance.

In many plant species, the time of day at which flowers open to permit pollination is tightly regulated. Proper time of flower opening, or Time of Day of Anther Appearance (TAA), may coordinate flowering opening with pollinator activity or may shift temperature sensitive developmental processes to cooler times of the day. The genetic mechanisms that regulate the timing of this process in cereal crops are unknown. To address this knowledge gap, it is necessary to establish a monocot model system that exhibits variation in TAA. Here, we examine the suitability of Setaria viridis, the model for C4 photosynthesis, for such a role. We developed an imaging system to monitor the temporal regulation of growth, flower opening time, and other physiological characteristics in Setaria. This system enabled us to compare Setaria varieties Ames 32254, Ames 32276, and PI 669942 variation in growth and daily flower opening time. We observed that TAA occurs primarily at night in these three Setaria accessions. However, significant variation between the accessions was observed for both the ratio of flowers that open in the day vs. night and the specific time of day where the rate is maximal. Characterizing this physiological variation is a requisite step toward uncovering the molecular mechanisms regulating TAA. Leveraging the regulation of TAA could provide researchers with a genetic tool to improve crop productivity in new environments.

Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases.

Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.

Bacteriophage 5' untranslated regions for control of plastid transgene expression.

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

Chloroplast transformation for engineering of photosynthesis.

Many efforts are underway to engineer improvements in photosynthesis to meet the challenges of increasing demands for food and fuel in rapidly changing environmental conditions. Various transgenes have been introduced into either the nuclear or plastid genomes in attempts to increase photosynthetic efficiency. We examine the current knowledge of the critical features that affect levels of expression of plastid transgenes and protein accumulation in transplastomic plants, such as promoters, 5' and 3' untranslated regions, RNA-processing sites, translation signals and amino acid sequences that affect protein turnover. We review the prior attempts to manipulate the properties of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through plastid transformation. We illustrate how plastid operons could be created for expression of the multiple genes needed to introduce new pathways or enzymes to enhance photosynthetic rates or reduce photorespiration. We describe here the past accomplishments and future prospects for manipulating plant enzymes and pathways to enhance carbon assimilation through plastid transformation.

Global and grain-specific accumulation of glycoside hydrolase family 10 xylanases in transgenic maize (Zea mays).

In planta expression of cell wall degrading enzymes is a promising approach for developing optimized biomass feedstocks that enable low-cost cellulosic biofuels production. Transgenic plants could serve as either an enzyme source for the hydrolysis of pretreated biomass or as the primary biomass feedstock in an autohydrolysis process. In this study, two xylanase genes, Bacillus sp. NG-27 bsx and Clostridium stercorarium xynB, were expressed in maize (Zea mays) under the control of two different promoters. Severe phenotypic effects were associated with xylanase accumulation in maize, including stunted plants and sterile grains. Global expression of these xylanases from the rice ubiquitin 3 promoter (rubi3) resulted in enzyme accumulation of approximately 0.01 mg enzyme per gram dry weight, or approximately 0.1% of total soluble protein (TSP). Grain-specific expression of these enzymes from the rice glutelin 4 promoter (GluB-4) resulted in higher-level accumulation of active enzyme, with BSX and XynB accumulating up to 4.0% TSP and 16.4% TSP, respectively, in shriveled grains from selected T0 plants. These results demonstrate the potential utility of the GluB-4 promoter for biotechnological applications. The phenotypic effects of xylanase expression in maize presented here demonstrate the difficulties of hemicellulase expression in an important crop for cellulosic biofuels production. Potential alternate approaches to achieve xylanase accumulation in planta without the accompanying negative phenotypes are discussed.

An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts.

Production of enzymes for lignocellulose hydrolysis in planta has been proposed as a lower-cost alternative to microbial production, with plastid transformation as a preferred method due to high foreign protein yields. An important regulator of chloroplast protein production is the downstream box (DB) region, located immediately downstream of the start codon. Protein accumulation can vary over several orders of magnitude by altering the DB region. Experiments in bacteria have suggested that these differences in protein accumulation may result from changes in translation efficiency, though the precise mechanism of DB function is not known. In this study, three DB regions were fused to the bglC ORF encoding a ß-glucosidase from the thermophilic bacterium Thermobifida fusca and inserted into the tobacco (Nicotiana tabacum) plastid genome. More than a two order of magnitude of difference in BglC protein accumulation was observed, dependent on the identity of the DB fusion. Differential transcript accumulation explained some the observed differences in protein accumulation, but in addition, less 3' degradation of bglC transcripts was observed in transgenic plants that accumulated the most BglC enzyme. Chloroplast-produced BglC was active against both pure cellobiose and against tobacco lignocellulose. These experiments demonstrate the potential utility of transplastomic plants as a vehicle for heterologous ß-glucosidase production for the cellulosic ethanol industry.

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants.

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10-60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, "secondary" recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene.

High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions.

The Thermobifida fusca cel6A gene encoding an endoglucanase was fused to three different downstream box (DB) regions to generate cel6A genes with 14 amino acid fusions. The DB-Cel6A fusions were inserted into the tobacco (Nicotiana tabacum cv. Samsun) chloroplast genome for protein expression. Accumulation of Cel6A protein in transformed tobacco leaves varied over approximately two orders of magnitude, dependent on the identity of the DB region fused to the cel6A open reading frame (ORF). Additionally, the DB region fused to the cel6A ORF affected the accumulation of Cel6A protein in aging leaves, with the most effective DB regions allowing for high level accumulation of Cel6A protein in young, mature, and old leaves, while Cel6A protein accumulation decreased with leaf age when less effective DB regions were fused to the cel6A ORF. In the most highly expressed DB-Cel6A construct, enzymatically active Cel6A protein accumulated at up to 10.7% of total soluble leaf protein (%TSP). The strategy used for high-level endoglucanase expression may be useful for expression of other cellulolytic enzymes in chloroplasts, ultimately leading to cost-effective heterologous enzyme production for cellulosic ethanol using transplastomic plants.

A sensitive and quantitative assay for normal PrP in plasma.

Transmissible spongiform encephalopathies can be transmitted by blood transfusion. The risk of spreading the disease among the human population could be mitigated with the implementation of a blood screening assay. We developed a two-antibody assay for PrP detection in plasma using the ORIGEN technology with a protocol modification to improve the limit of detection and to increase the sample volume assayed. In the standard 200 microL format, the assay had a detection limit of 7-10 pg of recombinant PrP and 3 pg in 1 mL final volume implementation. PrP concentration measured in normal and scrapie-infected hamster brains was 7.5+/-0.9 and 57.3+/-9.6 microg/g, respectively. After a concentration step with an immuno-affinity resin, plasma PrP(c) was detected by Western blot and its concentration was measured at 3.5+/-0.8 ng/mL. From these data and assuming that blood has the same specific infectivity as brain, we estimated the concentration of abnormal PrP in hamster-infected plasma to be 32 f g/mL. The assay also detected abnormal brain PrP spiked into plasma although the limit of detection was affected. This is a novel and sensitive assay for the detection of PrP in plasma that could be developed into a platform for a plasma-based TSE test.

Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco.

An inexpensive source of active cellulases is critical to efficient and cost-effective conversion of lignocellulosic biomass to ethanol. Transgenic plants expressing foreign cellulases are potential sources of cellulases for biomass conversion. A number of foreign proteins have been reported to accumulate to high levels when the transgene is incorporated into the chloroplast genome rather than into the nuclear genome. We developed plastid transformation vectors carrying two Thermobifida fusca thermostable cellulases, Cel6A and Cel6B, and expressed them in nicotine-free or nicotine-containing tobacco varieties following chloroplast transformation. We obtained homoplasmic tobacco plants expressing Cel6A or Cel6B. Maximum estimates of expression levels ranged from 2 to 4% of total soluble protein. Enzyme assays indicated that both Cel6A and Cel6B expressed in transplastomic tobacco were active in hydrolyzing crystalline cellulose. With further optimization, it may be feasible to produce bacterial cellulases in tobacco chloroplasts in large quantities.