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Non-apoptotic pioneer neutrophils initiate an endogenous swarming response in a zebrafish tissue injury model

Neutrophils are rapidly recruited to inflammatory sites where they are able to coordinate their migration to form clusters, a process termed neutrophil swarming. The factors which initiate neutrophil swarming are not understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study neutrophil migration, we demonstrate that neutrophil swarming is conserved in zebrafish immunity, sharing essential features with mammalian systems. We identified that one pioneer neutrophil was sufficient to induce neutrophil swarming after adopting a distinctive morphology at the wound site, followed by the coordinated migration of neutrophils to form a swarm. Using a FRET reporter of neutrophil apoptosis, we demonstrate that pioneer neutrophils do not undergo caspase-3 mediated apoptosis prior to swarming. These data provide some of the first evidence of endogenous neutrophil migration patterns prior to swarming and demonstrate that the zebrafish can be used to dissect the mechanisms modulating neutrophil swarm initiation.


39
Inflammation is the coordinated response of immune cells to invading pathogens or 40 endogenous danger signals. Sterile inflammation has evolved as a physiological response to 41 noxious stimuli including mechanical trauma, ischemia, toxins and antigens in the absence of 42 infection 1 . Neutrophils are one of the first responders to sterile inflammation, which rapidly 43 home to inflamed tissue within hours of injury. Within inflamed tissue, neutrophils carry out 44 specialised functions to destroy pathogens 2 and repair damage 3 , ultimately leading to the 45 restoration of tissue homeostasis. Neutrophils are recruited to an inflammatory stimulus 46 through a series of well-defined molecular events which lead to their extravasation from the 47 circulation into the tissue 4-6 . During their recruitment, neutrophils are primed by pro-48 inflammatory stimuli including growth factors, inflammatory cytokines and chemoattractants, 49 a process which increases responsiveness to activating agents and enhances neutrophil 50 function 7 . Within interstitial tissues, neutrophils are capable of integrating host-and pathogen-51 derived environmental signals, resulting in their polarisation and migration towards the 52 initiating inflammatory stimulus 8 . However, the precise mechanisms by which neutrophils 53 coordinate their migration and function within the complexity of inflamed interstitial tissue 54 remain to be understood. 55 Advances in intravital imaging have increased our understanding of the spatiotemporal 56 dynamics of neutrophil migration within interstitial tissue in vivo 9 . Neutrophils in the interstitium 57 coordinate their migration patterns to form clusters in several models of sterile-inflammation 58 and infection [9][10][11][12][13] . The parallels between these cellular behaviours and migration patterns seen 59 in insects has led to use of the term "swarming". A series of sequential phases leading to 60 neutrophil swarming have been described; the initial migration of 'pioneer' or 'scouting' 61 neutrophils proximal to the wound site (scouting) is followed by large scale synchronised 62 migration of neutrophils from distant regions (amplification) leading to neutrophil clustering 63 (stabilisation) and eventually resolution 9-12 . Communication between neutrophils during 64 swarming is complex. Many chemoattractants including lipid and proteins mediate the 65 response, with a dominant role for the lipid leukotriene B4 (LTB4) identified in vivo 10,11 . LTB4 66 produced by early responding neutrophils amplifies neutrophil tissue responses by signal relay 67 to more distant tissue regions 11,14 . Less is understood about the initiating signals required for 68 neutrophil attraction during the early stages of neutrophil swarming at sites of tissue damage. 69 Various chemoattractants from damaged cells and pathogens are present within an inflamed 70 tissue during the early stages of inflammation, making the functional dissection of the signals 71 required for neutrophil swarming challenging. The initial arrest and clustering of a small 72 7 of neutrophil swarms measured at the wound site during the 5 hour imaging period. Error bars 158 shown are mean ± SEM, n=5 experimental repeats. D. Proportion of neutrophil swarming 159 behaviour observed at the wound site within 5 hours following injury, n=5 experimental 160 repeats. E-F. Relay signalling through LTB4 is required for neutrophil recruitment. E. 161 Distance/time plot demonstrating the early recruitment of neutrophils proximal to the wound 162 site (<350μm) followed by the later recruitment of more distant neutrophils. F. CRISPR/Cas9-163 mediated knockdown of LTB4 signalling reduces late neutrophil recruitment. Neutrophil counts 164 at the wound site in control tyr crRNA injected larvae (black line), lta4h crRNA injected larvae 165 (grey dotted line), and blt1 crRNA injected larvae (black dotted line) at 3 and 6hpi. Error bars 166 shown are mean ± SEM. Groups were analysed using an ordinary one-way ANOVA and 167 adjusted using Tukey's multi comparison test. **p>0.008 n=45 from 3 independent 168 experiments.

Neutrophil swarms are initiated by a pioneer neutrophil with distinct morphology 202
The factors which initiate neutrophil swarming are not well defined. Neutrophil swarms in 203 mammals grow by large-scale migration towards 'pioneer neutrophils' in the context of both 204 sterile inflammation and infection, which likely release additional chemoattractants to initiate 205 the swarming response 9,11,12 . To understand whether a pioneer neutrophil is distinct to other 206 early responding neutrophils with a specialised capability to initiate a swarm, the migration 207 patterns of neutrophils in the time period preceding the swarming response were analysed by 208 reverse chronological tracking of neutrophil migration to persistent swarms ( Figure 3A). The 209 presence of one individual neutrophil with a distinct morphology was identified in the tissue 210 region which became the swarm centre in 100% of swarming events examined ( Figure 3B-E). 211 Based on its early recruitment and location at the swarm centre, this neutrophil is referred to 212 as the pioneer neutrophil. Prior to swarming, the pioneer neutrophil remained stationary in the 213 tissue region which became the swarm centre for on average 36 ± 7 minutes ( Figure 3B). 214 Pioneer neutrophils were rounded and non-motile, a distinct morphology which is illustrated 215 by their higher circularity index and lower displacement compared to scouting neutrophils 216 migrating at the wound site in the frame before swarming ( Figure 3C-E). Strikingly in 100% of 217 swarm initiation events examined, the pioneer neutrophil was the focal point of migration for 218 swarming neutrophils, whilst non-swarming neutrophils migrated randomly within the wound 219 region ( Figure 3D-E, Supplemental Movie 5). 220 the wound site is observed (frames 1-2) followed by the directed migration of swarming 232 neutrophils towards the pioneer, which is the focal point for migration (frames 3-5). The result 233 of migration is the aggregation of neutrophils to form swarms (frame 6). Tracks are coloured 234 by time where red corresponds to early and yellow corresponds to late arriving neutrophils. G. 235 Distance-time plot (DTP) of individual cell migration paths of swarming neutrophils (black 236 tracks) and non-swarming neutrophils at the wound site in the same time period (grey tracks). 237 Tracks are relative to pioneer neutrophil position; swarming neutrophils migrate to the pioneer 238 neutrophil whilst non-swarming neutrophils do not (n= 4 independent experiments). 239 Neutrophil swarming responses to tissue damage occur in three sequential stages 240 To determine the relationship between the pioneer neutrophil and swarm initiation, neutrophil 241 migration in the tail-fin at the entire population level was studied. Although there was variation 242 from fish-to-fish in timing, all swarms formed by: 1) the early recruitment of neutrophils to the 243 inflammatory site (scouting), 2) the behavioural change of a pioneer neutrophil at the wound 244 site (initiation), followed by 3) the directed migration of neutrophils to the pioneer to form 245 swarms (aggregation) (Figure 4, Supplemental Movie 6). Within minutes of injury, neutrophils 246 began directed migration to the wound site ( Figure 4A). This early scouting of neutrophils 247 lasted on average 88 ± 24 minutes and is consistent with reports in zebrafish 18 and mammalian 248 systems which describe the recruitment of neutrophils close to the inflammatory site in 249 response to chemoattractant gradients 9 . Swarm initiation began when the pioneer neutrophil 250 adopted its rounded, non-motile morphology having arrived at the wound site during the 251 scouting phase ( Figure 4B) and ended when the first neutrophil joined the swarm (on average 252 36 ± 7 minutes). During the aggregation phase, swarms developed through the directed 253 migration of neutrophils, which lasted on average 183 ± 25, or until the end of the imaging 254 period ( Figure 4C). As proof of concept, a non-biased approach was used to study pioneer 255 neutrophil migration. Pioneer neutrophils were tracked during the time period preceding the 256 start of swarming, where a change in pioneer neutrophil behaviour was observed, correlating 257 with the scouting and initiation phases ( Figure 4D-E). These stages provide consistent phases 258 with which to study pioneer neutrophil behaviour between larvae and are comparable to the 259 swarm stages reported in mammals 9,10 . 260

Pioneer neutrophils adopt a distinct morphology at the wound site 278
We next investigated whether the morphology observed in pioneer neutrophils prior to 279 swarming was distinct, or common, to all neutrophils upon arrival at the wound site. Tracks of 280 neutrophils migrating to the wound site during the scouting and the initiation phases were 281 extracted ( Figure 5A-B) and parameters which describe neutrophil motility including speed, 282 displacement and meandering index were analysed 15,16 . The speed, displacement and 283 meandering index of pioneer neutrophils were significantly reduced in the initiation phase 284 when compared to the scouting phase, whilst neutrophils migrating to the wound site within 285 the same tissue region did not display this behavioural change ( Figure 5C-E). These data 286 demonstrate that pioneer neutrophils display a distinct morphology at the wound site prior to 287 swarm formation, which is not seen in scouting neutrophils responding to chemoattractants 288 produced at the wound edge. Taken together, these findings suggest that within the complexity 289 of the inflamed tail-fin, specific guidance cues are produced from a single pioneer neutrophil 290 which enables neutrophils to coordinate their migration to form swarms. by the total length of the neutrophil track). Error bars are mean ± SEM. Groups were analysed 299 using a paired t-test *p<0.05 **p<0.01, n=5 independent experiments. 300

Pioneer neutrophils are not apoptotic prior to swarming 301
Cell death signalling has been implicated in neutrophil swarm initiation 9,11 , although the precise 302 signals and mode of cell death remain to be determined. The rounded, non-motile morphology 303 of pioneer neutrophils is characteristic of an apoptotic neutrophil phenotype previously 304 reported 24 . Furthermore apoptotic cells secrete "find-me" and "eat-me" signals to promote the 305 attraction of phagocytes for successful removal of apoptotic bodies 25 , therefore we 306 hypothesised that cell death signals released by apoptotic neutrophils could initiate the 307 swarming response. Neutrophil apoptosis can be studied using the transgenic Tg(mpx:CFP-308 DEVD-YFP)sh237 24 zebrafish line (known as mpx:FRET) which expresses a genetically 309 encoded Förster resonance energy transfer (FRET) 26 biosensor consisting of a caspase-3 310 cleavable DEVD sequence flanked by a CFP/YFP pair, under the neutrophil-specific mpx 311 promoter. A loss of FRET signal in this system provides a read out of apoptosis specifically in 312 neutrophils in vivo in real time. Analysis of pioneer neutrophils prior to swarming within the tail-313 fin ( Figure 6A) identified that despite the rounded, non-motile morphology observed in pioneer 314 neutrophils, a FRET signal was present during both the scouting and initiation phases in all 315 imaging studies where swarming was observed ( Figure 6B, Supplemental Movie 7, n=6 316 neutrophils from 5 experimental repeats). Furthermore, when an apoptotic event was detected 317 within the inflamed tail-fin, it was not followed by a neutrophil swarming response ( Figure 6C, 318 Supplemental Movie 8, n=2 neutrophils from 5 experimental repeats). These findings suggest 319 that neutrophil apoptosis does not initiate neutrophil swarming, and that despite their 320 morphology, pioneer neutrophils are not undergoing caspase-3 dependent apoptosis. Further 321 study of neutrophil behaviour in this model using transgenic reporters of cell death will enable 322 the dissection of the molecular cues which regulate swarm initiation. 323  Neutrophil responses to tissue injury are thought to occur in phases: the early recruitment of 361 neutrophils (referred to as 'scouting') is followed by the large scale synchronised migration of 362 neutrophils from distant regions (amplification), resulting in large scale tissue infiltration from 363 the bloodstream 9-11 . We demonstrate in our model that neutrophil response to tail fin 364 inflammation is bi-phasic; neutrophils proximal to the wound edge are recruited within minutes 365 following injury, whilst neutrophils from further away recruited between 2-6 hours following 366 injury. This is reminiscent of the bi-phasic neutrophil response to focal tissue damage 367 described in mice 9,11 . The time period of 6 hours required for recruitment in our system likely 368 reflects the difference in assays adopted between our study and mammalian studies; the 369 propagation of signals through the whole animal is required for neutrophil migration from tissue 370 niches in zebrafish whilst in mammals, neutrophils are injected proximal to the damage site. 371 In mammals, early neutrophil recruitment is modulated by signals released from damaged or 372 necrotic cells which are likely to be damage-associated molecular patterns (DAMPs). These 373 DAMPs include DNA, histones, interleukin-1α (IL-1α), N-formyl peptides and Adenosine 374 triphosphate (ATP) (reviewed in 27 ). These signals can be short-lived 17 , so the production of 375 longer term signals is required for sustained neutrophil recruitment 28 . LTB4 is a signal-relay 376 molecule which acts over long distances to promote neutrophil recruitment to formyl peptides 377 released from the centre of inflammatory sites 14 . We inhibited LTB4 signalling by targeting the 378 LTA4H enzyme or the LTB4 receptor using CRISPR/Cas9 and found neutrophil responses 379 were impaired only in the later stages of recruitment (3-6hpi). In zebrafish the CRISPR/Cas9 380 system is highly efficient, resulting in biallelic gene disruption in F0 zebrafish embryos which 381 allows for direct phenotypic analysis of injected animals 22 . Based on the expression of lta4h 382 and blt1 on neutrophils we propose that disruption of these genes will affect neutrophil 383 production and detection of LTB4. Our data agree with those from mice studies, showing that 384 the recruitment of neutrophils deficient in the LTB4 receptor is impaired only in neutrophils 385 distant to the focal tissue injury, whilst those proximal to the wound site are recruited like 386 wildtype neutrophils 11 . Evidence from zebrafish studies have demonstrated that following tail-387 fin transection, gradients of neutrophil chemoattractant signals are produced within minutes, 388 which extend up to 200µm into the tail fin epithelium as a concentration gradient 17 . Hydrogen 389 peroxide and chemokines such as CXCL8 are known to be important in neutrophil recruitment 390 in zebrafish larvae 17,29 . We therefore propose that the initial recruitment of neutrophils to the 391 wound site is dependent on the release of these chemoattractants, whilst signalling through 392 LTB4 is required to attract neutrophils at later stages from more distant tissue regions. 393 Factors which are likely to influence the swarm outcome include the size of the initiating tissue 394 damage, the presence of pathogens in the tissue, induction of secondary cell death and the 395 number of neutrophils initially recruited 19 . Linear tail-fin transection avoids creating a focal 396 source of neutrophil chemoattractants, migration towards which could mimic a swarming 397 response without the requirement for neutrophil-neutrophil signalling. Based on our findings 398 we propose that the formation of neutrophil swarms within a complex environment of diffusing 399 chemotactic gradients at the wound site would be dependent on intercellular signalling 400 between neutrophils. The zebrafish model therefore is a truly physiological system in which to 401 study the early events that determine the outcome of neutrophil swarming. Within the inflamed 402 tail-fin tissue, we found that neutrophil swarms developed around one individual neutrophil. 403 The single-cell resolution achieved in our study enabled us to make the striking observation 404 that this pioneer neutrophil adopted a distinct morphology at the wound site prior to swarming. 405 Other groups have found that within inflamed or infected interstitial tissue, the initial arrest of 406 a small number of 'pioneer' or 'scouting' neutrophils precedes a later influx of neutrophil 407 migration 11,12 . In these studies, it is unclear whether pioneer neutrophils are simply early 408 responding 'scouting' neutrophils, or if they have a specialised capacity for swarm initiation. 409 Based on our observations, we distinguished pioneer neutrophils from other scouting 410 neutrophils and propose that pioneer neutrophils have specialised functions required for 411 swarm initiation, whilst scouting neutrophils are simply early responders to chemoattractants 412 produced by damaged cells or pathogens at the inflammatory site. 413 Neutrophil swarming at the wound site in our system occurred in three distinct stages, which 414 are comparable to the sequential phases described in the swarming of neutrophils in 415 intravenous/ intradermal transfer models in mice 9,30 . Ng et al. describe a three phase cascade 416 of events to describe neutrophil migration towards laser induced or sterile needle induced 417 tissue injury 9 , which was further adapted into a five step attraction model 30 . In our linear tail-418 fin model we found that migration patterns leading to swarming shares features of both 419 models. During the scouting phase we observed the chemotactic movement of neutrophils 420 proximal to the wound site, sharing features with the scouting observed in mice and human 421 neutrophils 9,10 . The initial recruitment of neutrophils proximal to the wound site is common to 422 inflammation induced by infection or tissue injury, where these neutrophil 'scouts' are likely 423 responding to gradients of chemoattractants produced by damaged cells or pathogens 9,10 . 424 One pioneer neutrophil within the inflamed tail-fin was sufficient to initiate swarming in 425 zebrafish larvae, sharing function with the pioneer neutrophils essential for swarm initiation in 426 mice 30 . Due to the relatively few number of neutrophils present in zebrafish larvae (~300) in 427 comparison with the thousands (2-5x10 4 ) 9 injected into the mouse ear, we propose that 428 signals generated from just one pioneer neutrophil are sufficient to drive a swarming response 429 in our system. The pioneer behavioural change was observed during the swarm 'initiation' 430 phase. The initiation phase encapsulates the time period in which the pioneer neutrophil 431 adopted a rounded non-motile morphology at the wound site, until the first swarming neutrophil 432 makes contact. We propose that this stage is comparable to stage 2 'swarm amplification by 433 cell death' reported in mice 30 . Following its arrest, we observed directed migration of 434 neutrophils towards the pioneer during the aggregation phase which lasted until the end of the 435 imaging period in many larvae. The aggregation phase corresponds to the aggregation phase 436 reported by Lammermann 30 , and the cluster 'stabilisation' phase described by Ng 9 . The 437 parallels between the migration patterns leading swarming in zebrafish with those reported in 438 mice 9,30 and humans 10 suggests that the initiation of swarming is conserved between species. 439 Furthermore, these stages provide measurable time periods for the comparison of neutrophil 440 behaviour in future experiments to determine the signals released by pioneer neutrophils. 441 Based on the morphology of pioneer neutrophils we investigated whether neutrophil apoptosis 442 generated the chemoattractant signals required to initiate a swarming response within the tail-443 fin. Interestingly caspase-3 was intact during the swarm initiation phase, indicating that swarm 444 initiating pioneer neutrophils were not undergoing neutrophil apoptosis prior to swarming. Due 445 to the requirement for live imaging to study pioneer neutrophils prior to swarming, it was not 446 technically possible to confirm our results using staining assays such as TUNEL. However, 447 other studies have found that results using the mpx:FRET transgenic line recapitulate TUNEL 448 staining 24 , suggesting this is a reliable way to read out neutrophil apoptosis. The successful 449 application of the FRET transgenic reporter line to study apoptosis during swarm initiation 450 identifies that it is possible to study neutrophil swarm initiation in different reporter lines, which 451 will be useful in future to investigate other cell death signals important for swarm initiation. It 452 has been proposed that within the interstitium, neutrophils must prioritise 'superior' our study appeared to be viable prior to swarming, suggesting that lysis is not an initiating 471 factor in this model, although a programmed cell death process such as apoptosis or NETosis 472 is possible. Given that neutrophils respond to a multitude of chemoattractant signals, it is likely 473 that the release of multiple signals could be responsible for swarming behaviour. 474 Our findings in this study suggest that the zebrafish model of neutrophil swarming will be 475 extremely useful in dissecting the signalling which modulates early stages of neutrophil 476 swarming. Measuring and inhibiting intercellular signalling molecules is technically challenging 477 in vivo, posing significant barriers to dissecting the modulators of swarming at different stages. 478 Further elucidation of the nature of the pioneer neutrophil will require the development of new 479 technologies for the read-out of cell death phenomena and cytokine production in vivo. Using 480 a combination of transgenic zebrafish lines expressing cell-death read outs in neutrophils and 481 cell viability dyes, we will investigate pioneer neutrophil death as a potential mechanism for 482 swarm-initiation. Furthermore, the development of CRISPR interference technology and 483 neutrophil specific drivers of dead Cas9 by our group will enable us to inhibit genes of interest 484 in neutrophils specifically for loss-of-function studies, to identify the signals important in early 485 swarm initiation. These techniques will bypass limitations of other systems to allow the 486 dissection of early-swarming signals in vivo. 487 Understanding why swarms are initiated will be important for understanding the signals which 488 control the coordination of neutrophil migration within interstitial tissues. Our findings identify 489 that neutrophil swarm initiation at sites of tissue damage requires signals from one pioneer 490 neutrophil and that these signals can be dissected in future using the zebrafish model. To study neutrophils during inflammation Tg(mpx:EGFP)i114 (known as mpx:GFP) zebrafish 495 larvae were in-crossed. All zebrafish were raised in the Bateson Centre at the University of 496 Sheffield in UK Home Office approved aquaria and maintained following standard protocols 34 . 497 Tanks were maintained at 28°C with a continuous re-circulating water supply and a daily 498 light/dark cycle of 14/10 hours. All procedures were performed on embryos less than 5.2 dpf 499 which were therefore outside of the Animals (Scientific Procedures) Act, to standards set by 500 the UK Home Office. 501

Tail-fin transection assay 502
To induce an inflammatory response, zebrafish larvae at 2 or 3dpf were anaesthetised in 503 Tricaine (0.168 mg/ml; Sigma-Aldrich) in E3 media and visualised under a dissecting 504 microscope. Tail-fins were transected consistently using a scalpel blade (5mm depth, WPI) by 505 slicing immediately posterior to the circulatory loop, ensuring the circulatory loop remained 506 intact as previously described 18 . 507

Widefield microscopy of transgenic larvae 508
For neutrophil tracking experiments, injured 3dpf mpx:GFP larvae were mounted in a 1% low 509 melting point agarose solution (Sigma-Aldrich) containing 0.168 mg/ml tricaine immediately 510 following tail fin transection. Agarose was covered with 500μl of a clear E3 solution containing 511 0.168 mg/ml tricaine to prevent dehydration. Time lapse imaging was performed from 0.5-5 512 hours post injury with acquisition every 30 seconds using 10 z-planes were captured per larvae 513 over a focal range of 100μm using an Andor Zyla 5 camera (Nikon) and a GFP specific filter 514 with excitation at 488nm. Maximum intensity projections were generated by NIS elements 515 (Nikon) to visualise all 10 z-planes. 516

Confocal microscopy of transgenic larvae 517
For visualising neutrophil swarming at high magnification, larvae were mounted in a 1% low 518 melting point agarose solution (Sigma-Aldrich) containing 0.168 mg/ml tricaine for imaging 519 immediately after tail transection. Agarose was covered with 2000μl of clear E3 solution 520 containing 0.168 mg/ml tricaine to prevent dehydration. Imaging was performed from 30 521 minutes post injury using a 20x or 40x objective on an UltraVIEWVoX spinning disc confocal 522 laser imaging system (Perkin Elmer). Fluorescence for GFP was acquired using an excitation 523 wavelength of 488nm and emission was detected at 510nm, and fluorescence for mCherry 524 was acquired using 525nm emission and detected at 640nm. Images were processed using 525 Volocity™ software. 526

Tracking assays 527
Tracking of GFP labelled neutrophils was performed using NIS Elements (Version 4.3) with 528 an additional NIS elements tracking module. A binary layer was added to maximum intensity 529 projections to detect objects. Objects were smoothed, cleaned and separated to improve 530 accuracy. A size restriction was applied where necessary to exclude small and large objects 531 which did not correspond to individual neutrophils. 532

Distance-time plots 533
For wound plots the distances from the wound were obtained by processing neutrophil tracks 534 under the assumption that the tail fin wound is a straight line parallel to the x-axis of the 535 greyscale image. Neutrophil tracking data was extracted from NIS elements and imported into 536 MatLab software. For distance to pioneer plots the pioneer centre was set as a reference point 537 and tracking was performed to determine neutrophil distance to the reference point. Tracks 538 were extracted from NIS elements and plotted manually using GraphPad Prism version 7.0.

Microinjection of SygRNA® into embryos 557
A 1nl drop of SygRNA®:Cas9 protein complex was injected into mpx:GFP embryos at the one-558 cell stage. Embryos were collected at the one cell stage and injected using non-filament glass 559 capillary needles (Kwik-Fil TM Borosilicate Glass Capillaries, World Precision Instruments 560 (WPI), Herts, UK). RNA was prepared in sterile Eppendorf tubes. A graticule was used to 561 measure 0.5nl droplet sizes to allow for consistency of injections. Injections were performed 562 under a dissecting microscope attached to a microinjection rig (WPI) and a final volume of 1nl 563 was injected. 564

Genotyping and melting curve analysis 565
Site-specific mutations were detected using High Resolution Melting (HRM) Analysis which 566 can reliably detect CRISPR/Cas9 induced indels in embryos 37,38 . Genomic DNA extraction 567 was performed on larvae at 2dpf. Larvae were placed individually in 0.2ml PCR tubes in 90µl 568 50mM NaOH and boiled at 95° for 20 minutes. 10µl Tris-HCL ph8 was added as a reaction 569 buffer and mixed thoroughly. Gene specific primers were designed using the Primer 3 web 570 tool (http://primer3.ut.ee/). Sequences were as follows lta4h_fw: 571 CGTGTAGGTTAAAATCCATTCGCA lta4h_rev: GAGAGCGAGGAGAAGGAGCT blt1_fw: 572 GTCTTCTCTGGACCACCTGC blt1_rev: ACACAAAAGCGATAACCAGGA. HRM analysis 573 (Bio-Rad) PCR reactions were made with 5µl Sybr™ Green master mix (Thermo Fisher), 0.5µl 574 of each primer (10µM), 1µl gDNA and 3µl water to make a final reaction volume of 10µl. PCR 575 reactions were performed in a LightCycler instrument (Bio-Rad) using 96-well plates. The two-576 step reaction protocol was as follows: 95 °C for 2 min, followed by 35 cycles of 95 °C for 10 577 seconds, 58° for 30 seconds, 72° for 20 seconds. The second stage of the protocol was 95 °C 578 for 30 seconds, 60 °C for 60 seconds, 65 °C for 10 seconds. The temperature then increased 579 by 0.02 °C/s until 95 °C for 10 seconds. Melt curves were analysed using Bio-Rad software 580 version 1.2. Succesful detection of CRISPR/Cas9 induced indels is illustrated in supplemental 581 figure 6. Mutagenesis frequencies of 91% and 88% were detected for lta4h and blt1 582 respectively. 583

Staphylococcus aureus preparation 584
Staphylococcus aureus strain SH1000 pMV158mCherry was used for all experiments 39 . An 585 overnight bacterial culture was prepared by growing 1cfu of SH1000 pMV158mCherry in 586