Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination.

Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.

Extreme drought impacts have been underestimated in grasslands and shrublands globally.

Climate change is increasing the frequency and severity of short-term (~1 y) drought events-the most common duration of drought-globally. Yet the impact of this intensification of drought on ecosystem functioning remains poorly resolved. This is due in part to the widely disparate approaches ecologists have employed to study drought, variation in the severity and duration of drought studied, and differences among ecosystems in vegetation, edaphic and climatic attributes that can mediate drought impacts. To overcome these problems and better identify the factors that modulate drought responses, we used a coordinated distributed experiment to quantify the impact of short-term drought on grassland and shrubland ecosystems. With a standardized approach, we imposed ~a single year of drought at 100 sites on six continents. Here we show that loss of a foundational ecosystem function-aboveground net primary production (ANPP)-was 60% greater at sites that experienced statistically extreme drought (1-in-100-y event) vs. those sites where drought was nominal (historically more common) in magnitude (35% vs. 21%, respectively). This reduction in a key carbon cycle process with a single year of extreme drought greatly exceeds previously reported losses for grasslands and shrublands. Our global experiment also revealed high variability in drought response but that relative reductions in ANPP were greater in drier ecosystems and those with fewer plant species. Overall, our results demonstrate with unprecedented rigor that the global impacts of projected increases in drought severity have been significantly underestimated and that drier and less diverse sites are likely to be most vulnerable to extreme drought.

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.

Expanding the flexibility of base editing for high-throughput genetic screens in bacteria.

Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.

Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.

CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.

Dryland sensitivity to climate change and variability using nonlinear dynamics.

Primary productivity response to climatic drivers varies temporally, indicating state-dependent interactions between climate and productivity. Previous studies primarily employed equation-based approaches to clarify this relationship, ignoring the state-dependent nature of ecological dynamics. Here, using 40 y of climate and productivity data from 48 grassland sites across Mongolia, we applied an equation-free, nonlinear time-series analysis to reveal sensitivity patterns of productivity to climate change and variability and clarify underlying mechanisms. We showed that productivity responded positively to annual precipitation in mesic regions but negatively in arid regions, with the opposite pattern observed for annual mean temperature. Furthermore, productivity responded negatively to decreasing annual aridity that integrated precipitation and temperature across Mongolia. Productivity responded negatively to interannual variability in precipitation and aridity in mesic regions but positively in arid regions. Overall, interannual temperature variability enhanced productivity. These response patterns are largely unrecognized; however, two mechanisms are inferable. First, time-delayed climate effects modify annual productivity responses to annual climate conditions. Notably, our results suggest that the sensitivity of annual productivity to increasing annual precipitation and decreasing annual aridity can even be negative when the negative time-delayed effects of annual precipitation and aridity on productivity prevail across time. Second, the proportion of plant species resistant to water and temperature stresses at a site determines the sensitivity of productivity to climate variability. Thus, we highlight the importance of nonlinear, state-dependent sensitivity of productivity to climate change and variability, accurately forecasting potential biosphere feedback to the climate system.

Reintroducing bison results in long-running and resilient increases in grassland diversity.

The widespread extirpation of megafauna may have destabilized ecosystems and altered biodiversity globally. Most megafauna extinctions occurred before the modern record, leaving it unclear how their loss impacts current biodiversity. We report the long-term effects of reintroducing plains bison (Bison bison) in a tallgrass prairie versus two land uses that commonly occur in many North American grasslands: 1) no grazing and 2) intensive growing-season grazing by domesticated cattle (Bos taurus). Compared to ungrazed areas, reintroducing bison increased native plant species richness by 103% at local scales (10 m2) and 86% at the catchment scale. Gains in richness continued for 29 y and were resilient to the most extreme drought in four decades. These gains are now among the largest recorded increases in species richness due to grazing in grasslands globally. Grazing by domestic cattle also increased native plant species richness, but by less than half as much as bison. This study indicates that some ecosystems maintain a latent potential for increased native plant species richness following the reintroduction of native herbivores, which was unmatched by domesticated grazers. Native-grazer gains in richness were resilient to an extreme drought, a pressure likely to become more common under future global environmental change.

Sheet Models for Methylaluminoxane (MAO) Activators? A Theoretical Case Study involving rac-Me2Si(η5-C9H6)2Zr (SBIZr) Complexes.

Activation of SBIZrMe2 or SBIZrMeCl and a sheet model for an active component of hydrolytic MAO, (MeAlO)16(Me3Al)6, (16,6) has been studied by DFT. Contact ion-pair formation occurs through the intermediacy of SBIZrMe(Cl) or SBIZrMe2 reacting with sheet 16,6 to furnish SBIZrMe-µ-X(MeAlO)16(Me3Al)6 (2, X=Me, Cl). Contact ion-pairs 2 would be in equilibrium with heterodinuclear catalyst precursors [SBIZrMe2AlMe2][(MeAlO)16(Me3Al)6X] (3 (X=Me, Cl) through reversible binding of Me3Al at higher Al : Zr ratios. Calculations show that formation of ion-pairs 3 from contact ion-pairs 2 is more favourable for the SBIZr compared with the parent Cp2Zr complexes. TD-DFT calculations were conducted on relevant SBIZr complexes to relate the results to earlier spectroscopic studies of catalyst activation using UV-Vis spectroscopy. Finally, propene insertion into ion-pairs 2, SBIZrMe-µ-MeB(C6F5)3 (6) and [SBIZrMe][B(C6F5)4] (7) was studied at M06-2X/TZVP level of theory. These studies suggest that contact ion-pairs 2 are significantly less reactive towards insertion than 6 or 7, in disagreement with experiment.

Heterogeneity promotes resilience in restored prairie: Implications for the environmental heterogeneity hypothesis.

Enhancing resilience in formerly degraded ecosystems is an important goal of restoration ecology. However, evidence for the recovery of resilience and its underlying mechanisms require long-term experiments and comparison with reference ecosystems. We used data from an experimental prairie restoration that featured long-term soil heterogeneity manipulations and data from two long-term experiments located in a comparable remnant (reference) prairie to (1) quantify the recovery of ecosystem functioning (i.e., productivity) relative to remnant prairie, (2) compare the resilience of restored and remnant prairies to a natural drought, and (3) test whether soil heterogeneity enhances resilience of restored prairie. We compared sensitivity and legacy effects between prairie types (remnant and restored) and among four prairie sites that included two remnant prairie sites and prairie restored under homogeneous and heterogeneous soil conditions. We measured sensitivity and resilience as the proportional change in aboveground net primary productivity (ANPP) during and following drought (sensitivity and legacy effects, respectively) relative to average ANPP based on 4 pre-drought years (2014-2017). In nondrought years, total ANPP was similar between remnant and restored prairie, but remnant prairie had higher grass productivity and lower forb productivity compared with restored prairie. These ANPP patterns generally persisted during drought. The sensitivity of total ANPP to drought was similar between restored and remnant prairie, but grasses in the restored prairie were more sensitive to drought. Post-drought legacy effects were more positive in the restored prairie, and we attributed this to the more positive and less variable legacy response of forb ANPP in the restored prairie, especially in the heterogeneous soil treatment. Our results suggest that productivity recovers in restored prairie and exhibits similar sensitivity to drought as in remnant prairie. Furthermore, creating heterogeneity promotes forb productivity and enhances restored prairie resilience to drought.

Updates on coding in dermatology.

Correct coding is an important component of effective dermatology practice management. Over the past several years there have been updates to many commonly used codes within dermatology. This review highlights many of these updates, such as: the skin biopsy codes have been subdivided to reflect the different biopsy techniques. The definition of complex linear repairs has been updated and clarified. Outpatient and inpatient evaluation and management visits have new coding guidelines to determine level of care. Dermatopathology consultation codes have been updated and category III codes related to digital pathology have been created. Understanding the details and nuances of each of these categories of codes is vital to ensuring appropriate coding is performed.

Modeling diffusion-weighted imaging lesion expansion between 2 and 24 h after endovascular thrombectomy in acute ischemic stroke.

PURPOSE: Diffusion-weighted imaging (DWI) lesion expansion after endovascular thrombectomy (EVT) is not well characterized. We used serial diffusion-weighted magnetic resonance imaging (MRI) to measure lesion expansion between 2 and 24 h after EVT. METHODS: In this single-center observational analysis of patients with acute ischemic stroke due to large vessel occlusion, DWI was performed post-EVT (< 2 h after closure) and 24-h later. DWI lesion expansion was evaluated using multivariate generalized linear mixed modeling with various clinical moderators. RESULTS: We included 151 patients, of which 133 (88%) had DWI lesion expansion, defined as a positive change in lesion volume between 2 and 24 h. In an unadjusted analysis, median baseline DWI lesion volume immediately post-EVT was 15.0 mL (IQR: 6.6-36.8) and median DWI lesion volume 24 h post-EVT was 20.8 mL (IQR: 9.4-66.6), representing a median change of 6.1 mL (IQR: 1.5-17.7), or a 39% increase. There were no significant associations among univariable models of lesion expansion. Adjusted models of DWI lesion expansion demonstrated that relative lesion expansion (defined as final/initial DWI lesion volume) was consistent across eTICI scores (0-2a, 0.52%; 2b, 0.49%; 2c-3, 0.42%, p = 0.69). For every 1 mL increase in lesion volume, there was 2% odds of an increase in 90-day mRS (OR: 1.021, 95%CI [1.009, 1.034], p < 0.001). CONCLUSION: We observed substantial lesion expansion post-EVT whereby relative lesion expansion was consistent across eTICI categories, and greater absolute lesion expansion was associated with worse clinical outcome. Our findings suggest that alternate endpoints for cerebroprotectant trials may be feasible.

A Microfabricated, Flow-Driven Grinding Mill for Mechanical Cell Lysing.

This paper presents the design, microfabrication, and demonstration of a novel microfluidic grinding mill for the lysis of the dinoflagellate, Alexandrium, a neurotoxin-producing genus of algae that is responsible for red tide and paralytic shellfish poisoning. The mill consists of a high-speed, hydrodynamically driven microrotor coupled to a micro grinding mill that lyses robust algal cells by mechanical abrasion with single-pass efficiencies as high as 97%. These efficiencies are comparable to, or better than, current mechanical and chemical lysing methods without adding complications associated with harsh chemical additives that can interfere with subsequent downstream bioanalysis. Release of cytoplasm from lysed algae was confirmed using polymerase chain reaction (PCR) amplification of Alexandrium DNA using dinoflagellate primers.

As above, not so below: Long-term dynamics of net primary production across a dryland transition zone.

Drylands are key contributors to interannual variation in the terrestrial carbon sink, which has been attributed primarily to broad-scale climatic anomalies that disproportionately affect net primary production (NPP) in these ecosystems. Current knowledge around the patterns and controls of NPP is based largely on measurements of aboveground net primary production (ANPP), particularly in the context of altered precipitation regimes. Limited evidence suggests belowground net primary production (BNPP), a major input to the terrestrial carbon pool, may respond differently than ANPP to precipitation, as well as other drivers of environmental change, such as nitrogen deposition and fire. Yet long-term measurements of BNPP are rare, contributing to uncertainty in carbon cycle assessments. Here, we used 16 years of annual NPP measurements to investigate responses of ANPP and BNPP to several environmental change drivers across a grassland-shrubland transition zone in the northern Chihuahuan Desert. ANPP was positively correlated with annual precipitation across this landscape; however, this relationship was weaker within sites. BNPP, on the other hand, was weakly correlated with precipitation only in Chihuahuan Desert shrubland. Although NPP generally exhibited similar trends among sites, temporal correlations between ANPP and BNPP within sites were weak. We found chronic nitrogen enrichment stimulated ANPP, whereas a one-time prescribed burn reduced ANPP for nearly a decade. Surprisingly, BNPP was largely unaffected by these factors. Together, our results suggest that BNPP is driven by a different set of controls than ANPP. Furthermore, our findings imply belowground production cannot be inferred from aboveground measurements in dryland ecosystems. Improving understanding around the patterns and controls of dryland NPP at interannual to decadal scales is fundamentally important because of their measurable impact on the global carbon cycle. This study underscores the need for more long-term measurements of BNPP to improve assessments of the terrestrial carbon sink, particularly in the context of ongoing environmental change.

Accounting for herbaceous communities in process-based models will advance our understanding of "grassy" ecosystems.

Grassland and other herbaceous communities cover significant portions of Earth's terrestrial surface and provide many critical services, such as carbon sequestration, wildlife habitat, and food production. Forecasts of global change impacts on these services will require predictive tools, such as process-based dynamic vegetation models. Yet, model representation of herbaceous communities and ecosystems lags substantially behind that of tree communities and forests. The limited representation of herbaceous communities within models arises from two important knowledge gaps: first, our empirical understanding of the principles governing herbaceous vegetation dynamics is either incomplete or does not provide mechanistic information necessary to drive herbaceous community processes with models; second, current model structure and parameterization of grass and other herbaceous plant functional types limits the ability of models to predict outcomes of competition and growth for herbaceous vegetation. In this review, we provide direction for addressing these gaps by: (1) presenting a brief history of how vegetation dynamics have been developed and incorporated into earth system models, (2) reporting on a model simulation activity to evaluate current model capability to represent herbaceous vegetation dynamics and ecosystem function, and (3) detailing several ecological properties and phenomena that should be a focus for both empiricists and modelers to improve representation of herbaceous vegetation in models. Together, empiricists and modelers can improve representation of herbaceous ecosystem processes within models. In so doing, we will greatly enhance our ability to forecast future states of the earth system, which is of high importance given the rapid rate of environmental change on our planet.

Ionization of Cp2 ZrMe2 and Lewis Bases by Methylaluminoxane: Computational Insights.

The interactions of the Lewis bases CO, octamethyltrisiloxane (OMTS) and 2,2'-bipyridine (bipy) with a sheet model for the principal activator (MeAlO)16 (Me3 Al)6 (16,6) in hydrolytic methylaluminoxane (MAO) were investigated by DFT. These studies reveal that OMTS and bipy form adducts with Me3 Al prior to methide abstraction by 16,6 to form the ion-pairs [Me2 Al(κ2 -L)][16,6] (5: L=OMTS, 6: L=bipy, [16,6]- =[(MeAlO)16 (Me3 Al)6 Me]- ) while CO simply binds to a reactive edge site without ionization. The binding and activation of Cp2 ZrMe2 with 16,6 to form both neutral adducts 1 Cp2 ZrMe2 ⋅16,6 and contact ion-pairs 4 and 7, both with formula [Cp2 ZrMe][µ-Me(MeAlO)16 (Me3 Al)6 ], featuring terminal and chelated MAO-anions, respectively was studied by DFT. The displacement of the anion with either excess Cp2 ZrMe2 or Me3 Al was also studied, forming outer-sphere ion-pairs [(Cp2 ZrMe)2 µ-Me][16,6] (2) and [Cp2 Zr(µ-Me)2 AlMe2 ][16,6] (3). The theoretical NMR spectra of these species were compared to experimental spectra of MAO and Cp2 ZrMe2 and found to be in good agreement with the reported data and assignments. These studies confirm that 16,6 is a very suitable model for the activators present in MAO but highlight the difficulty in accurately calculating thermodynamic quantities for molecules in this size regime.

Cages versus Sheets: A Critical Comparison in the Size Range Expected for Methylaluminoxane (MAO).

New cage models (MeAlO)n (Me3 Al)m (n=16, m=6 or 7) isomeric with previously reported sheet models for the principle activator found in hydrolytic MAO (h-MAO) are compared at M06-2X and MN15 levels of theory using density functional theory with respect to their thermodynamic stability. Reactivity of the neutrals or corresponding anions with formula [(MeAlO)16 (Me3 Al)6 Me]- towards chlorination, and loss of Me3 Al is explored while reactivity of the neutrals towards formation of contact- and outer-sphere ion pairs from Cp2 ZrMe2 and Cp2 ZrMeCl is examined. The results suggest on balance that a cage model for this activator is less consistent with experiment than an isomeric sheet model, although the latter are more stable based on free energy.

Soil nematode assemblages respond to interacting environmental changes.

Multi-factor experiments suggest that interactions among environmental changes commonly influence biodiversity and community composition. However, most field experiments manipulate only single factors. Soil food webs are critical to ecosystem health and may be particularly sensitive to interactions among environmental changes that include soil warming, eutrophication, and altered precipitation. Here, we asked how environmental changes interacted to alter soil nematode communities in a northern Chihuahuan Desert grassland. Factorial manipulations of nitrogen, winter rainfall, and nighttime warming matched predictions for regional environmental change. Warming reduced nematode diversity by 25% and genus-level richness by 32%, but declines dissipated with additional winter rain, suggesting that warming effects occurred via drying. Interactions between precipitation and nitrogen also altered nematode community composition, but only weakly affected total nematode abundance, indicating that most change involved reordering of species abundances. Specifically, under ambient precipitation, nitrogen fertilizer reduced bacterivores by 68% and herbivores by 73%, but did not affect fungivores. In contrast, under winter rain addition, nitrogen fertilization increased bacterivores by 95%, did not affect herbivores, and doubled fungivore abundance. Rain can reduce soil nitrogen availability and increase turnover in the microbial loop, potentially promoting the recovery of nematode populations overwhelmed by nitrogen eutrophication. Nematode communities were not tightly coupled to plant community composition and may instead track microbes, including biocrusts or decomposers. Our results highlight the importance of interactions among environmental change stressors for shaping the composition and function of soil food webs in drylands.

Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.

CRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a 'trigger' RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5' end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.

Non-Contact Irreversible Electroporation in the Esophagus With a Wet Electrode Approach.

Our objective was to develop a technique for performing irreversible electroporation (IRE) of esophageal tumors while mitigating thermal damage to the healthy lumen wall. We investigated noncontact IRE using a wet electrode approach for tumor ablation in a human esophagus with finite element models for electric field distribution, joule heating, thermal flux, and metabolic heat generation. Simulation results indicated the feasibility of tumor ablation in the esophagus using an catheter mounted electrode immersed in diluted saline. The ablation size was clinically relevant, with substantially lesser thermal damage to the healthy esophageal wall when compared to IRE performed by placing a monopolar electrode directly into the tumor. Additional simulations were used to estimate ablation size and penetration during noncontact wet-electrode IRE (wIRE) in the healthy swine esophagus. A novel catheter electrode was manufactured and wIRE evaluated in seven pigs. wIRE was performed by securing the device in the esophagus and using diluted saline to isolate the electrode from the esophageal wall while providing electric contact. Computed tomography and fluoroscopy were performed post-treatment to document acute lumen patency. Animals were sacrificed within four hours following treatment for histologic analysis of the treated esophagus. The procedure was safely completed in all animals; post-treatment imaging revealed intact esophageal lumen. The ablations were visually distinct on gross pathology, demonstrating full thickness, circumferential regions of cell death (3.52 ± 0.89 mm depth). Acute histologic changes were not evident in nerves or extracellular matrix architecture within the treatment site. Catheter directed noncontact IRE is feasible for performing penetrative ablations in the esophagus while avoiding thermal damage.

Resolving the Dust Bowl paradox of grassland responses to extreme drought.

During the 1930s Dust Bowl drought in the central United States, species with the C3 photosynthetic pathway expanded throughout C4-dominated grasslands. This widespread increase in C3 grasses during a decade of low rainfall and high temperatures is inconsistent with well-known traits of C3 vs. C4 pathways. Indeed, water use efficiency is generally lower, and photosynthesis is more sensitive to high temperatures in C3 than C4 species, consistent with the predominant distribution of C3 grasslands in cooler environments and at higher latitudes globally. We experimentally imposed extreme drought for 4 y in mixed C3/C4 grasslands in Kansas and Wyoming and, similar to Dust Bowl observations, also documented three- to fivefold increases in C3/C4 biomass ratios. To explain these paradoxical responses, we first analyzed long-term climate records to show that under nominal conditions in the central United States, C4 grasses dominate where precipitation and air temperature are strongly related (warmest months are wettest months). In contrast, C3 grasses flourish where precipitation inputs are less strongly coupled to warm temperatures. We then show that during extreme drought years, precipitation-temperature relationships weaken, and the proportion of precipitation falling during cooler months increases. This shift in precipitation seasonality provides a mechanism for C3 grasses to respond positively to multiyear drought, resolving the Dust Bowl paradox. Grasslands are globally important biomes and increasingly vulnerable to direct effects of climate extremes. Our findings highlight how extreme drought can indirectly alter precipitation seasonality and shift ecosystem phenology, affecting function in ways not predictable from key traits of C3 and C4 species.