Natural and Engineered Guide RNA-directed Transposition with CRISPR-Associated Tn7-like Transposons.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.

Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons.

CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways: one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Multiple mechanisms allow functionally distinct guide RNAs for transposition: a conventional system capable of acquiring guide RNAs to new plasmid and phage targets and a second providing long-term memory for access to chromosomal sites upon entry into a new host. Guide RNAs are privatized to be recognized only by the transposon-adapted system via sequence specialization, mismatch tolerance, and selective regulation to avoid toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals promising avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.

Assembly of the Tn7 targeting complex by a regulated stepwise process.

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.

Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition.

CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition.

Structural basis of transposon end recognition explains central features of Tn7 transposition systems.

Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.

Novel mechanisms of diversity generation in Acinetobacter baumannii resistance islands driven by Tn7-like elements.

Mobile genetic elements play an important role in the acquisition of antibiotic and biocide resistance, especially through the formation of resistance islands in bacterial chromosomes. We analyzed the contribution of Tn7-like transposons to island formation and diversification in the nosocomial pathogen Acinetobacter baumannii and identified four separate families that recognize different integration sites. One integration site is within the comM gene and coincides with the previously described Tn6022 elements suggested to account for the AbaR resistance island. We established Tn6022 in a heterologous E. coli host and confirmed basic features of transposition into the comM attachment site and the use of a novel transposition protein. By analyzing population features within Tn6022 elements we identified two potential novel transposon-encoded diversification mechanisms with this dynamic genetic island. The activities of these diversification features were confirmed in E. coli. One was a novel natural gain-of-activity allele that could function to broaden transposition targeting. The second was a transposon-encoded hybrid dif-like site that parasitizes the host dimer chromosome resolution system to function with its own tyrosine recombinase. This work establishes a highly active Tn7-like transposon that harnesses novel features allowing the spread and diversification of genetic islands in pathogenic bacteria.

Discovery and characterization of novel type I-D CRISPR-guided transposons identified among diverse Tn7-like elements in cyanobacteria.

CRISPR-Cas defense systems have been naturally coopted for guide RNA-directed transposition by Tn7 family bacterial transposons. We find cyanobacterial genomes are rich in Tn7-like elements, including most of the known guide RNA-directed transposons, the type V-K, I-B1, and I-B2 CRISPR-Cas based systems. We discovered and characterized an example of a type I-D CRISPR-Cas system which was naturally coopted for guide RNA-directed transposition. Multiple novel adaptations were found specific to the I-D subtype, including natural inactivation of the Cas10 nuclease. The type I-D CRISPR-Cas transposition system showed flexibility in guide RNA length requirements and could be engineered to function with ribozyme-based self-processing guide RNAs removing the requirement for Cas6 in the heterologous system. The type I-D CRISPR-Cas transposon also has naturally fused transposase proteins that are functional for cut-and-paste transposition. Multiple attributes of the type I-D system offer unique possibilities for future work in gene editing. Our bioinformatic analysis also revealed a broader understanding of the evolution of Tn7-like elements. Extensive swapping of targeting systems was identified among Tn7-like elements in cyanobacteria and multiple examples of convergent evolution, including systems targeting integration into genes required for natural transformation.

Widespread CRISPR-derived RNA regulatory elements in CRISPR-Cas systems.

CRISPR-cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR-cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR-cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8, or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.

Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM.

CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.

Transposition into replicating DNA occurs through interaction with the processivity factor.

The bacterial transposon Tn7 directs transposition into actively replicating DNA by a mechanism involving the transposon-encoded protein TnsE. Here we show that TnsE physically and functionally interacts with the processivity factor of the DNA replication machinery in vivo and in vitro. Our work establishes an in vitro TnsABC+E transposition reaction reconstituted from purified proteins and target DNA structures. Using the in vitro reaction we confirm that the processivity factor specifically reorders TnsE-mediated transposition events on target DNAs in a way that matches the bias with active DNA replication in vivo. The TnsE interaction with an essential and conserved component of the replication machinery, and a DNA structure reveals a mechanism by which Tn7, and probably other elements, selects target sites associated with DNA replication.

Perceptions of High-Intensity Interval Training Among People With Spinal Cord Injury: A Mixed-Methods Analysis.

This mixed-method project investigated how people with spinal cord injury perceive high-intensity interval training (HIIT). Using a recumbent hand cycle, 11 active men and 9 active women with spinal cord injury or related disease participated in a single HIIT and moderate-intensity continuous training (MICT) session. Following exercise, participants completed surveys assessing enjoyment, self-efficacy, and outcome expectations. Ten participants were randomly selected to participate in a semistructured interview to assess perceptions toward HIIT. Quantitative survey data revealed that participants trended toward enjoying HIIT over MICT (p = .06) with similar levels of self-efficacy and outcome expectations toward HIIT and MICT (p > .05). Qualitative data revealed that participants believed HIIT would enhance long-term physical and self-evaluative outcomes; several barriers emerged that could prevent widespread adoption among the general population with spinal cord injury. Results support HIIT as a viable exercise option, although research should begin exploring ways to remove HIIT-related barriers that people with spinal cord injury may encounter.

Vulnerabilities on the lagging-strand template: opportunities for mobile elements.

Mobile genetic elements have the ability to move between positions in a genome. Some of these elements are capable of targeting one of the template strands during DNA replication. Examples found in bacteria include (a) Red recombination mediated by bacteriophage λ, (b) integration of group II mobile introns that reverse splice and reverse transcribe into DNA, (c) HUH endonuclease elements that move as single-stranded DNA, and (d) Tn7, a DNA cut-and-paste transposon that uses a target-site-selecting protein to target transposition into certain forms of DNA replication. In all of these examples, the lagging-strand template appears to be targeted using a variety of features specific to this strand. These features appear especially available in certain situations, such as when replication forks stall or collapse. In this review, we address the idea that features specific to the lagging-strand template represent vulnerabilities that are capitalized on by mobile genetic elements.

The effectiveness of vigorous training on cardiorespiratory fitness in persons with spinal cord injury: a systematic review and meta-analysis.

DESIGN: Systematic review and meta-analysis. BACKGROUND AND PURPOSE: Traditional forms of upper-body moderate intensity exercise consistently provide little cardiovascular benefits for persons with spinal cord injury (PwSCI). Explorations of new training methods are vital to improve cardiovascular fitness among PwSCI. This study sought to evaluate the effectiveness of vigorous training on cardiorespiratory fitness in PwSCI. METHODS: Database search through PubMed, Web of Science, Scopus, SportDiscus, and Cumulative Index of Nursing and Allied Health Literature (CINAHL) was conducted from the databases' inception to November 2020 to identify relevant exercise studies with PwSCI. Two independent reviewers screened articles for inclusion. Data were extracted from included studies and methodological quality evaluated. RESULTS: Sixteen trials (eight pre-post trials and eight controlled trials [CTs]) with a total of 145 participants were analyzed. Results from pre-post studies revealed significant improvements in cardiorespiratory fitness following high-intensity interval training (HIIT) (Peak Oxygen Uptake [VO2peak], standardized mean difference [SMD] = 0.81; 95% CI 0.23-1.39; P < 0.01 and Peak Power Output [PPO], SMD = 0.91; 95% CI 0.32-1.5; P < 0.01) and circuit resistance training (CRT) (VO2peak, MD = 0.38; 95% CI 0.19-0.57; P < 0.01 and PPO, MD = 20.17; 95% CI 8.26-32.08; P < 0.01). Meta-analysis of CTs did not demonstrate significant improvements in cardiorespiratory fitness following vigorous training interventions in comparison to lower intensity training interventions. CONCLUSION: Evidence from HIIT and CRT interventions suggest benefits for cardiovascular functions; however, vigorous training was not more beneficial than other forms of endurance training. More CTs are needed to better understand the effectiveness of vigorous training on cardiorespiratory fitness in PwSCI.

Effects of various walking intensities on leg muscle fatigue and plantar pressure distributions.

BACKGROUND: Physical activity may benefit health and reduce risk for chronic complications in normal and people with diabetes and peripheral vascular diseases. However, it is unclear whether leg muscle fatigue after weight-bearing physical activities, such as brisk walking, may increase risk for plantar tissue injury. In the literature, there is no evidence on the effect of muscle fatigue on plantar pressure after various walking intensities. The objectives of this study were to investigate the effects of various walking intensities on leg muscle fatigue and plantar pressure patterns. METHODS: A 3 × 2 factorial design, including 3 walking speeds (1.8 (slow and normal walking), 3.6 (brisk walking), and 5.4 (slow running) mph) and 2 walking durations (10 and 20 min) for a total of 6 walking intensities, was tested in 12 healthy participants in 3 consecutive weeks. The median frequency and complexity of electromyographic (EMG) signals of tibialis anterior (TA) and gastrocnemius medialis (GM) were used to quantify muscle fatigue. Fourier transform was used to compute the median frequency and multiscale entropy was used to calculate complexity of EMG signals. Peak plantar pressure (PPP) values at the 4 plantar regions (big toe, first metatarsal head, second metatarsal head, and heel) were calculated. RESULTS: Two-way ANOVA showed that the walking speed (at 1.8, 3.6, 5.4 mph) significantly affected leg muscle fatigue, and the duration factor (at 10 and 20 min) did not. The one-way ANOVA showed that there were four significant pairwise differences of the median frequency of TA, including walking speed of 1.8 and 3.6 mph (185.7 ± 6.1 vs. 164.9 ± 3.0 Hz, P = 0.006) and 1.8 and 5.4 mph (185.7 ± 6.1 vs. 164.5 ± 5.5 Hz, P = 0.006) for the 10-min duration; and walking speed of 1.8 and 3.6 mph (180.0 ± 5.9 vs. 163.1 ± 4.4 Hz, P = 0.024) and 1.8 and 5.4 mph (180.0 ± 5.9 vs. 162.8 ± 4.9 Hz, P = 0.023) for the 20-min duration. The complexity of TA showed a similar trend with the median frequency of TA. The median frequency of TA has a significant negative correlation with PPP on the big toe ( r = -0.954, P = 0.003) and the first metatarsal head ( r = -0.896, P = 0.016). CONCLUSIONS: This study demonstrated that brisk walking and slow running speeds (3.6 and 5.4 mph) cause an increase in muscle fatigue of TA compared to slow walking speed (1.8 mph); and the increased muscle fatigue is significantly related to a higher PPP.

Gait and Balance Assessments using Smartphone Applications in Parkinson's Disease: A Systematic Review.

Gait dysfunctions and balance impairments are key fall risk factors and associated with reduced quality of life in individuals with Parkinson's Disease (PD). Smartphone-based assessments show potential to increase remote monitoring of the disease. This review aimed to summarize the validity, reliability, and discriminative abilities of smartphone applications to assess gait, balance, and falls in PD. Two independent reviewers screened articles systematically identified through PubMed, Web of Science, Scopus, CINAHL, and SportDiscuss. Studies that used smartphone-based gait, balance, or fall applications in PD were retrieved. The validity, reliability, and discriminative abilities of the smartphone applications were summarized and qualitatively discussed. Methodological quality appraisal of the studies was performed using the quality assessment tool for observational cohort and cross-sectional studies. Thirty-one articles were included in this review. The studies present mostly with low risk of bias. In total, 52% of the studies reported validity, 22% reported reliability, and 55% reported discriminative abilities of smartphone applications to evaluate gait, balance, and falls in PD. Those studies reported strong validity, good to excellent reliability, and good discriminative properties of smartphone applications. Only 19% of the studies formally evaluated the usability of their smartphone applications. The current evidence supports the use of smartphone to assess gait and balance, and detect freezing of gait in PD. More studies are needed to explore the use of smartphone to predict falls in this population. Further studies are also warranted to evaluate the usability of smartphone applications to improve remote monitoring in this population.Registration: PROSPERO CRD 42020198510.

Targeted transposition with Tn7 elements: safe sites, mobile plasmids, CRISPR/Cas and beyond.

Transposon Tn7 is notable for the control it exercises over where transposition events are directed. One Tn7 integration pathways recognizes a highly conserved attachment (att) site in the chromosome, while a second pathway specifically recognizes mobile plasmids that facilitate transfer of the element to new hosts. In this review, I discuss newly discovered families of Tn7-like elements with different targeting pathways. Perhaps the most exciting examples are multiple instances where Tn7-like elements have repurposed CRISPR/Cas systems. In these cases, the CRISPR/Cas systems have lost their canonical defensive function to destroy incoming mobile elements; instead, the systems have been naturally adapted to use guide RNAs to specifically direct transposition into these mobile elements. The new families of Tn7-like elements also include a variety of novel att sites in bacterial chromosomes where genome islands can form. Interesting families have also been revealed where proteins described in the prototypic Tn7 element are fused or otherwise repurposed for the new dual activities. This expanded understanding of Tn7-like elements broadens our view of how genetic systems are repurposed and provides potentially exciting new tools for genome modification and genomics. Future opportunities and challenges to understanding the impact of the new families of Tn7-like elements are discussed.

Recruitment of CRISPR-Cas systems by Tn7-like transposons.

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages.

A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), who together establish guidelines that define, in part, that the presence of Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24-48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.

Exploring the Influence of Wheelchair-User Interface and Personal Characteristics on Ischial Tuberosity Peak Pressure Index and Gradient in Elite Wheelchair Basketball Players.

This pilot study investigated the relationship between personal and wheelchair factors on skin pressures at the ischial tuberosity in wheelchair basketball players. Seventeen wheelchair basketball players (7 male and 10 female) were evaluated during static and dynamic propulsive conditions while peak pressure index and peak pressure gradient were recorded with an interface pressure mat. The results showed that greater seat dump angles and backrest heights were negatively associated with the peak pressure index. Therapeutic cushion use was moderately associated with a reduced peak pressure gradient. Higher-class players used chair configurations associated with augmented pressure; however, classification status alone was not associated with pressure magnitude. Body mass index was negatively correlated with the static peak pressure gradient at levels approaching significance (p < .10). In conclusion, greater seat dump angles and backrest heights may provide pressure relief, whereas greater body mass index and therapeutic cushion use may reduce pressure gradients.

Climate and fishing drive regime shifts in consumer-mediated nutrient cycling in kelp forests.

Globally, anthropogenic pressures are reducing the abundances of marine species and altering ecosystems through modification of trophic interactions. Yet, consumer declines also disrupt important bottom-up processes, like nutrient recycling, which are critical for ecosystem functioning. Consumer-mediated nutrient dynamics (CND) is now considered a major biogeochemical component of most ecosystems, but lacking long-term studies, it is difficult to predict how CND will respond to accelerating disturbances in the wake of global change. To aid such predictions, we coupled empirical ammonium excretion rates with an 18-year time series of the standing biomass of common benthic macroinvertebrates in southern California kelp forests. This time series of excretion rates encompassed an extended period of extreme ocean warming, disease outbreaks, and the abolishment of fishing at two of our study sites, allowing us to assess kelp forest CND across a wide range of environmental conditions. At their peak, reef invertebrates supplied an average of 18.3 ± 3.0 µmol NH4 +  m-2  hr-1 to kelp forests when sea stars were regionally abundant, but dropped to 3.5 ± 1.0 µmol NH4 +  m-2  hr-1 following their mass mortality due to disease during a prolonged period of extreme warming. However, a coincident increase in the abundance of the California spiny lobster, Palinurus interupptus (Randall, 1840), likely in response to both reduced fishing and a warmer ocean, compensated for much of the recycled ammonium lost to sea star mortality. Both lobsters and sea stars are widely recognized as key predators that can profoundly influence community structure in benthic marine systems. Our study is the first to demonstrate their importance in nutrient cycling, thus expanding their roles in the ecosystem. Climate change is increasing the frequency and severity of warming events, and rising human populations are intensifying fishing pressure in coastal ecosystems worldwide. Our study documents how these projected global changes can drive regime shifts in CND and fundamentally alter a critical ecosystem function.