Plasmodium falciparum population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. Despite high genetic diversity, we observe pronounced fine-scale spatial and temporal parasite genetic structure. Clusters of near-clonal infections on Pemba indicate persistent local transmission with limited parasite importation, presenting an opportunity for local elimination efforts. Furthermore, we observe an admixed parasite population on Unguja and detect a substantial fraction (2.9%) of significantly related infection pairs between Zanzibar and the mainland, suggesting recent importation. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and provides actionable insights for prioritizing malaria elimination efforts.Zanzibar has made significant progress toward malaria elimination, but recent stagnation requires novel approaches. We developed a highly multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug-resistance loci, and successfully sequenced 290 samples from five districts covering both main islands. Here, we elucidate fine-scale  Sequencing malaria parasites from low density infections in small amounts of dried blood is important for large-scale genomic surveillance. Here, the authors develop and validate a highly multiplexed droplet digital PCR-based amplicon deep sequencing assay and apply it to data from Zanzibar, Tanzania. Zanzibar, an archipelago and semi-autonomous region of the United Republic of Tanzania with its two main islands, Unguja and Pemba, has made great progress towards elimination of malaria in the last two decades. Malaria prevalence is now estimated to be less than 5% by PCR2, mainly as a result of the continued use of long-lasting insecticidal nets (LLIN), indoor residual spraying (IRS), and artemisinin-based combination therapies (ACTs)3.Despite the intensification of control interventions and renewed efforts to eliminate malaria, it remains one of the world’s most widespread and deadly infectious diseases, causing 247 million cases and killing more than 600,000 people in 20214. RACD includes screening by rapid diagnostic test (RDT) and treatment of household members of passively detected index cases at health facilities. However, despite considerable efforts, malaria elimination in Zanzibar remains elusive. There are likely several reasons for this: firstly, despite the increasingly focal nature of residual transmission in Zanzibar7, there is a substantial reservoir of asymptomatic infections in the community3. Many of these infections are low-density below the detection limit of RDTs9, making it challenging to identify and treat them. Secondly, despite the implementation of strong vector control measures, there is ongoing local transmission due to residual vector capacity3. Lastly, Zanzibar is highly connected to mainland Tanzania where malaria transmission remains substantially higher in certain areas12. Thus, even if local transmission has been reduced to very low levels, parasite importation through human travel might be a concern and obstacle to local elimination as long as the environment remains receptive13.Since 2012, the Zanzibar Malaria Elimination Program (ZAMEP) has implemented reactive case detection (RACD) to better target residual foci of transmission14. It can support the design of effective interventions by monitoring the spread of drug resistance markers17, assessing transmission intensity18, identifying foci of sustained transmission 20, estimating connectivity between parasite populations21, and distinguishing local from imported cases19. Parasite genetic data can identify genetically isolated parasite populations with limited gene flow between them, which might be preferred targets for local elimination by targeted control measures22. Further, it is crucial to monitor markers of malaria drug resistance. There is increasing evidence of parasites with markers of artemisinin partial resistance circulating in sub-Saharan Africa, including mainland Tanzania, Rwanda and Uganda, threatening the long-term effectiveness of ACT regimens, the current first-line treatment in Zanzibar26.Achieving elimination of malaria in Zanzibar requires a better understanding of why and where importation occurs, as well as the relative contribution of importation to sustaining local parasite populations. Malaria parasite genetic data provides information that is complementary to clinical and epidemiological data28. Targeted amplicon sequencing (AmpSeq) of the most informative regions in the genome allows for deep and consistent sequence coverage29. AmpSeq offers very high detectability of minority clones as low as 1% frequency in polyclonal infections29, which are frequent even in pre-elimination settings30. By targeting short, highly diverse microhaplotype loci that contain multiple SNPs and exhibit multiallelic rather than biallelic diversity, discriminatory power can be increased33. To date, many studies utilizing this method have only targeted one or a few genomic loci to gain information on diversity of infections, drug resistance, or selection36. Only recently, there have been efforts to extend these methods to large multiplexed AmpSeq panels covering numerous genetically diverse loci  and markers of drug-resistance, as pioneered by Tessema et al.32 and others33. Increasing the number of diverse loci can provide a higher resolution comparison of infections at the population level as well as pairwise relatedness inference at the individual level33.Novel amplicon sequencing-based genotyping methods allow typing of parasites at high resolution and sensitivity and provide a highly detailed picture of the parasite population structure37. It ranges from zero for completely unrelated individuals to one for identical clones. Identity-by-descent (IBD)-based methods allow estimating the probability that two alleles at a given locus in the genome are identical due to descent38. This provides a metric at finer spatiotemporal scales  for characterizing changes and measuring connectivity in parasite populations than classical population genetic measures of population diversity and divergence 21, on timescales relevant to malaria elimination efforts39. While many IBD-based methods use whole genome sequencing (WGS) data and are limited to monoclonal infections or to biallelic loci41, a recently developed approach allows to infer the level of shared common ancestry  between polyclonal infections from unphased multiallelic data such as microhaplotype data generated by AmpSeq42. Measures such as population genetic diversity, linkage disequilibrium, or multiplicity of infection (MOI) might be useful as surrogate markers of changes in transmission intensity30.Various genomic indices yield information relevant for malaria genomic epidemiology. Genetic relatedness is a recombination-based measure of recent shared ancestryP. falciparum in island settings potentially closely linked to mainland areas with higher transmission analyzed a limited number of samples and typed only few genomic markers44. Limited genomic data is available from the Zanzibar archipelago and its parasite connectivity to the mainland45. It is unclear whether additional infections identified through RACD are directly related to clinical index cases. A thorough genetic characterization of circulating parasites on the archipelago is crucial to elucidate travel patterns, and to identify within-country and cross-border parasite connectivity.Previous studies that have examined the population structure of 35 to characterize the parasite population structure and map antimalarial resistance profiles in Zanzibar, and define relatedness of parasites. The assay was optimized for high sensitivity to allow typing of asymptomatic, low-density samples collected as dried blood spot (DBS) on filter papers, which are a practical sample type for large-scale epidemiological studies. Here, we utilize multiplexed AmpSeq designed to quantify differentiation and relatedness between samples. We evaluate the parasite population diversity in Zanzibar and use this information to assess the relatedness between clinical index cases and the asymptomatic cases surrounding them identified by RACD, and to quantify relatedness of and connectivity between parasites on Zanzibar and mainland Tanzania and Kenya.We developed a novel high-throughput multiplexed AmpSeq method targeting highly diverse microhaplotypes and drug resistance lociWe developed and optimized a novel 2-step multiplex ddPCR-based assay for even amplification and detection of minority clones and validated the assay on a range of 3D7 whole blood and DBS controls. For control samples, the assay generated a high number of reads per sample and per locus after trimming low quality reads for both, whole blood samples and DBS samples  between the number of markers successfully genotyped and parasite density, with samples genotyped at ≥10 loci having significantly higher geometric mean parasite densities compared to samples genotyped at 9 loci or less . At densities ≥5 parasites/µL, 99.2% (241/243) samples had data for ≥10 loci. High coverage (median 98.6%) in samples with ≥10 parasites/µL was achieved  with a high number of unique alleles    , as well as higher MOI in individuals who reported recent travel   of infections in Zanzibar were polyclonal with a mean MOI of 1.85 (range 1–8) across districts Fig. . The gen01) Fig. . Correla01) Fig. . Overall73) Fig. . We alsoG′′ST and Jost’s D) identified high genetic differentiation of two districts on Pemba, Micheweni and Chake Chake, from the other districts . Similarly, inbreeding determined by mLD was higher in samples from Micheweni and Chake Chake, indicating the circulation of more closely related haplotypes in these populations  by pairwise identity by descent (IBD) using Dcifer. Expected sharing is 0.50 for full siblings and 0.25 for half-siblings with unrelated parents46. Among all pairs of parasites from Zanzibar, mean IBD was 0.037. We found the overall distribution of pairwise IBD to be heavy-tailed, with the majority of samples being unrelated and a tail of very highly related samples . We found a decay in genetic relatedness with increasing spatial distance in Zanzibar , indicating high relatedness of index cases and secondary cases. We also compared mean IBD and the proportion of related infections on different administrative units, by years, and by travel history , indicating that a proportion of the parasite population is maintained through the dry season and contributes to malaria cases the following season. We also found a 1.3-fold higher mean IBD among non-travelers vs. among travelers to mainland Tanzania and Kenya , possibly due to subtle population structure differences between Zanzibar and the mainland.We estimated the genetic relatedness of all pairs of samples , with the majority of within shehia related pairs found on Pemba . Similarly, the odds of finding related infections were significantly higher within RACD clusters than between RACD clusters , and within households than between households .Of the 1703 significantly related pairs we found 1335/40,432 (3.3%) pairs between shehias  related at the level of full siblings or closer (IBD ≥ 0.5), including 119 (0.28%) highly related sample pairs (IBD ≥ 0.9). These highly related pairs were found more frequently within the same shehia than between different shehias  and published genomes from mainland Tanzania (n = 80) and Kenya (n = 35). We specifically chose sites that represent travel destinations reported by the Zanzibari study participants . Three pairs with IBD > 0.5 were identified between three mainland samples and two individuals from Zanzibar  and central Africa  than to samples from west Africa and southeast Asia Fig. . Howeverpfdhfr , pfdhps , pfmdr1 (codons 86 and 184), pfmdr2 (codons 484 and 492), and pfk13   being particularly prevalent . We also observed a high number of the pfmdr1 N86-184F haplotype . We found no obvious spatial pattern for any of the drug resistance alleles.Focusing on 74 Zanzibar samples that were identified as monoclonal infections with complete allele calls for all seven drug resistance loci, we measured the prevalence of multidrug-resistant parasites and explored their spatial distribution . In line with the expectation of more frequent parasite importation on Unguja, parasite diversity was higher than on Pemba, and no population structure was observed in those highly admixed parasite populations, suggesting that importation from mainland Tanzania and Kenya play an important role. In contrast, several clonal or near-clonal outbreaks were observed in Micheweni and Chake Chake districts in Pemba, indicative of lower levels of importation from outside. This was further supported by increased genetic differentiation and higher levels of inbreeding in those two districts.We have applied a novel, multiplexed ddPCR-amplicon sequencing protocol targeting 28 microhaplotypes loci to provide a high-resolution view of parasite population structure and genetic relatedness of parasites in Zanzibar. We were able to capture information from polyclonal infections (60% of the study population) by using multiallelic loci, and to obtain robust results from low-density asymptomatic infections collected as DBS in the frame of routine RACD procedures Although commonly believed that 42 allowed for the incorporation of data from all parasites detected in infections. Despite high genetic diversity, we were able to extract useful signals of recent transmission created by recombination and co-transmission of multiple parasites and elucidate fine-scale population structure in Zanzibar, providing evidence of ongoing local transmission. Many malaria cases observed in two districts (Micheweni and Chake Chake) on Pemba appeared to be due to local transmission, evidenced by the strong fine-scale spatial structure in the genetic data. This was further supported by classical measures of genetic differentiation . Our genomic data corroborated previous spatio-temporal analyses on routinely collected data in Zanzibar from 2015 to 2020 that identified Micheweni district as a hotspot area with greater and more consistent local transmission than other districts55. We showed that parasites from individuals living closer together  were more highly related than more distant infection pairs. Infection pairs from within the same RACD clusters were more related than between different RACD clusters, indicating that index and additional secondary cases detected during the RACD investigation are often directly linked.Estimation of pairwise genetic relatedness using an IBD-based approach to infer the degree of shared ancestry between polyclonal infections from unphased multiallelic data56. Indeed, presumptive treatment of household members of malaria cases has been suggested to reduce transmission in Zanzibar, while testing and treating neighbors of malaria cases was shown to only have minimal additional impact on transmission13. The results of our study and previous findings from the RADZEC project8 have led ZAMEP to actively consider these approaches to enhance local elimination efforts. ZAMEP has initiated the process of developing standard operating procedures and piloting for reactive focal MDA. On the other hand, despite a signal of predominantly local transmission at smaller scales (within tens of kilometers), we also found that parasite populations in Zanzibar remain highly connected between geographically more distant districts (over tens to hundreds of kilometers), likely driven by human movement. We identified a substantial number of infection pairs at the relatedness level of half-siblings or more (IBD ≥ 0.25), potentially representing imported cases and providing evidence for recent, and most likely ongoing, genetic exchange between Zanzibar and the mainland. This is also supported by the finding that infected individuals detected during RACD most often reported recent travel to a district in mainland Tanzania that exhibits a very high malaria transmission risk57. While systems that target malaria infections and transmission at small scale can usefully eliminate local foci, efforts to eliminate malaria in Zanzibar will crucially depend on the reduction of ongoing importation of infections from mainland Tanzania58. Targeted efforts in Zanzibar over the past decade maintained incidence on the islands at low levels but a further reduction of transmission in both Zanzibar and mainland Tanzania will be necessary to eventually achieve elimination in Zanzibar58.Malaria elimination programs need to target interventions at local scales that are tailored to the drivers of the local transmission situation. More isolated populations with lower parasite diversity and little population movement, such as those identified in Micheweni and Chake Chake districts, would appear amenable to focal control interventions. In comparably isolated foci of local transmission, such efforts are expected to be more sustainable as these areas are less likely to experience regular reintroduction of parasites from other areas. Targeting such areas with a combination of reactive focal MDA and reactive vector control is expected to have a complementary effect and may be more effective than RACD alone, as shown in a trial in Namibia20. Even though parasite populations between Zanzibar and regions on the mainland of Tanzania remain genetically almost indistinguishable, we find subtle differences between the islands, and some districts. Pairwise IBD sharing revealed that travelers were significantly more related to isolates from mainland Tanzania and Kenya than non-travelers. We were even able to identify differences between travelers vs. non-travelers at the district level on Pemba. However, most of these relationships are far too remote to be attributed to the period covered by the travel history and more likely represent the presence of subtle population structure between Zanzibar and the mainland. Overall, this suggests a high degree of gene flow between Zanzibar and the mainland and likely presents a significant obstacle to reaching elimination on the archipelago.Travelers did not cluster separately Fig.  and therWhile denser sampling at sites frequently visited by travelers from Zanzibar might yield further highly related infections, genomic data alone likely is insufficient to differentiate imported infections from those locally transmitted, at such a fine geographical scale with little population genetic differentiation as is the case between Zanzibar and the mainland. We did not collect parasites outside of Zanzibar as part of our study, thus relying on publicly available genomes from the mainland, possibly precluding more fine-scale evaluations of connectivity between Zanzibar and the mainland. Further, sampling of parasites was linked to routine RACD by ZAMEP and hence not spatially uniform across geographic locations. Among the 290 samples successfully genotyped, the proportion of samples from within the same households varies from 25 to 66%, with no clear differences between Unguja and Pemba. While this data is broadly reflective of the occurrence of cases in the study districts, spatial uncertainty remains in areas with fewer notified index cases and in districts not included in this study.Our panel was able to distinguish parasites from southeast Asia, east Africa, and west Africa, confirming the validity of the microhaplotype panel to differentiate samples by continent of origin, though not by country. As expected, the isolates from Zanzibar fell in the east Africa cluster. Estimates of relatedness clearly showed that the Zanzibar isolates are more closely related to samples from east Africa and central Africa than to samples from west Africa. However, they are more highly related within Zanzibar than between any other country.59. In Zanzibar, frequencies of known drug-resistance alleles are similar to previous reports in East Africa, including Zanzibar59. Mutations associated with resistance to sulfadoxine-pyrimethamine (SP) in both the pfdhfr  and pfdhps (K540E) genes were observed at high frequencies, with parasites carrying all four mutations being particularly prevalent. We found high rates of the pfmdr1 N86 wildtype and 184 F mutant alleles, both of which are associated with decreased sensitivity to lumefantrine60. The pfmdr1 86Y mutation and Y184 wildtype alleles, conferring resistance to amodiaquine61, were observed at lower frequencies. The recently reported polymorphism in pfmdr2 T484I associated with artemisinin partial resistance alleles in Southeast Asia63 was not found. However, the pfmdr2 I492V mutation was prevalent, which is also suggested to be involved in artemisinin partial resistance63. Our study did not identify any known pfk13 artemisinin resistance-conferring mutations, suggesting that parasites in Zanzibar remain sensitive to the current first-line treatment ASAQ. The recent identification of pfk13 mutants  in mainland Tanzania23 and neighboring countries Rwanda26 and Uganda25, and the high prevalence of mutations in other drug resistance markers found in this study highlights the need for continued monitoring. Of note, the recently described mutations in the pfk13 gene found in Rwanda, Uganda, and mainland Tanzania were not covered by our panel.Drug resistance is a major threat to malaria control. The first-line ACT in Zanzibar has been artesunate/amodiaquine (ASAQ) since 2003, plus single, low-dose primaquine, which to date shows high efficacy in ZanzibarvarATS assay with primers targeting approximately 20 copies per genome64, and using a direct PCR without prior DNA extraction8. This approach is more sensitive than typing of a single-copy marker after DNA extraction65.Our assay performed well on low-density DBS (≥5 parasites/µL) without the need of sWGA pre-amplification prior to the first PCR. The ddPCR-amplicon sequencing approach yielded high read numbers across 35 loci and overall coverage of all markers in samples as low as 10 parasites/µL. More even amplification might be achieved by varying primer concentrations  to account for amplification rate differences among loci. It was expected that not all qPCR-positive samples could be successfully sequenced as initial parasite screening was done using the multi-copy In conclusion, our multiplex AmpSeq panel allowed us to determine MOI, parasite genetic diversity, population structure, genetic relatedness, and to monitor genetic markers associated with drug resistance on the Zanzibar archipelago. Our data presents the most comprehensive genomic assessment of malaria in an island setting closely linked to nearby areas of higher transmission. Our finding of isolated populations on Pemba provides information on fine-scale genetic structure of parasites directly relevant to urgent programmatic needs of the malaria elimination program in Zanzibar.4. The samples used in this study were collected between May 19, 2017, and October 31, 2018, during a rolling cross-sectional survey as part of the ReActive case Detection in Zanzibar: Effectiveness and Cost (RADZEC) study. In the two study years, ZAMEP reported a mean annual parasite incidence (API) per 1000 population of 2.6 , as compared to an API of 106 reported by the mainland NMCP66. The survey covered five districts  on both Unguja and Pemba, Zanzibar  were screened by direct amplification of P. falciparum DNA from 5 × 3 mm DBS punches and quantified using ultra-sensitive varATS qPCR as previously described8, resulting in a total of 518 positive samples.The 2022 census reported a population of 1,346,332 in Unguja and 543,441 in Pemba. The islands are highly connected to mainland Tanzania by two airports  and three main seaports Fig. . In addibar Fig. . SamplesEthical approval for this study was obtained from the Zanzibar Medical Research Ethics Committee (ZAMREC/0001/February/17), the Institutional Review Boards of Tulane University (Study Number: 993573) and of the Ifakara Health Institute (IHI/IRB/No: 003 – 2017), the Ethics Commission of North-western and Central Switzerland (EKNZ Reg-2017-00162), and the University of Notre Dame Institutional Review Board .P. falciparum positive DBS was extracted with the NucleoMag Blood 200 µL kit (Macherey-Nagel) using an optimized protocol for DNA extraction from whole 50 μL DBS, as previously described65. Briefly, 240 µL lysis buffer MBL1 and 620 µL PBS were added to each 50 μL DBS and incubated at 94 °C for 30 min. Samples were cooled, 40 µL Proteinase K was added and incubated at 60 °C and 300 rpm for 1 h. The amount of binding buffer MBL2 was adjusted to 800 µL, buffer MBL4 was omitted, and the protocol was followed according to the manufacturer’s recommendations. DNA was eluted in 50 µL buffer MBL5 and stored at −20 °C until use.Field-collected DBS were stored with desiccant at room temperature until processing. DNA from all 518 E) and 7 drug resistance loci distributed across the genome  AmpSeq protocol, targeting 28 microhaplotypes with high values for expected heterozygosity  according to the manufacturer’s recommendations.A secondary indexing PCR was performed in a total reaction volume of 50 µL containing 15 µL of purified primary PCR product, 300 nM of each Illumina TruSeq i5/i7 barcode primers , using filter criteria defined and explained in ref. 29. Briefly, sequencing reads were demultiplexed by sample and amplicon. Overlapping paired end reads were merged via the vsearch v2.22.1 package67 and clustered via the Swarm v2 package68. Nucleotide positions of the amplicons were only considered for haplotype calling, if they had a known SNP in PlasmoDB (https://plasmodb.org), or if they had a novel SNP that was dominant (>0.5 mismatch rate to the reference sequence) and was supported by at least two samples or replicates. A mismatch rate of >0.5 to the reference sequence indicates a potential novel SNP found on the dominant clone or a clone with a within-host frequency of >0.5 at MOI >1. For single clone infections a mismatch rate of 1 is expected. Chimeric reads, singletons, and reads containing an insertion or deletion were excluded. Haplotype calling from the remaining reads required a within-host haplotype frequency of ≥1% and a minimum coverage of ≥10 reads per haplotype in ≥2 samples or replicates. To further reduce false positive haplotype calls, each haplotype had to be confirmed by both replicates of a sample.The targeted amplicon deep sequencing data was analyzed using the P. falciparum whole blood mixture of 105 parasites/μL blood. This mixture was then further diluted with uninfected human whole blood to obtain serial dilutions with parasite densities of 104, 103, 102 and 10 parasites/μL blood. Similarly, we also generated mock DBS by spotting 50 µL mock whole blood mixtures on Whatman 3MM filter paper  at parasite densities of 103, 102 and 10 parasites/μL blood. DNA was extracted using the NucleoMag Blood 200 µL kit (Macherey-Nagel).We generated mock samples from parasite culture lines 3D7 and FCB. Parasite cultures were mixed with uninfected human whole blood to generate a mock 5 parasites/μL) were mixed at 60:40%  and 80:20% (4:1) ratios to mimic polyclonal field isolates. The ratios were serially diluted to 103, 102 and 10 parasites/μL. Furthermore, we included previously described mixed strain controls of 3D7:HB329 with a relative abundance of 2% (50:1) and 1% (100:1) for the minority clone (HB3) at a density of 103 parasites/μL.We also generated mixed-strain controls for minority clone detection. 3D7 and FCB strains at similar parasite density .We compared coverage of markers, fold-difference between the highest number of reads and lowest number of reads per sample, and the proportion of reads lost after trimming of low-quality reads between ddPCR and conventional PCR using E) using the formula, n is the number of genotyped samples and pi is the frequency of the ith allele in the population. MOI and HE were compared by district, island, year, and travel history. We calculated the probability  that two clones share all 28 haplotypes based on all apparent single-clone infections by concatenating the 28 microhaplotypes into one multi-locus haplotype for each monoclonal sample with data in all 28 microhaplotypes (n = 28) and for 27 microhaplotypes . HE for 28 microhaplotypes combined in all single clone infections was 0.9915, and 0.9921 for 27 microhaplotypes. Thus, the probability that two unrelated clones cannot be distinguished, and such an infection is falsely counted as single clone infection, is less than 1%. Data was visualized using ggplot2.Within-host diversity was determined using multiplicity of infection (MOI), defined as the highest number of alleles detected by at least two of the 28 microhaplotype loci. Population genetic diversity was estimated from the 28 microhaplotype loci by expected heterozygosity  using Bayesian clustering package rmaverick v 1.1.071, under the admixture model with 10,000 burn-in iterations, 2000 sampling iterations and 20 thermodynamic rungs. We estimated genetic differentiation  between districts using Jost’s D72 and G′′ST73, a modified version of Hedrick’s G′ST that corrects for a bias that stems from sampling a limited number of populations. Briefly, Jost’s D and G′′ST were calculated using the formulas: HT and HS are the overall and the subpopulation heterozygosity, respectively, and n is the number of sampled populations. The values of Jost’s D and G′′ST range from 0 (no genetic differentiation between populations) to 1 (complete differentiation between populations). As a measure of inbreeding in each population we computed the multilocus linkage disequilibrium (mLD) using LIAN version 3.7, applying a Monte Carlo test with 100,000 permutations74. Monoclonal and polyclonal infections  with complete haplotypes were included. All analyses were performed using the 28 microhaplotypes only, unless otherwise noted.We evaluated geographic clustering by discriminant analysis of principal components (DAPC) using the Dcifer, an IBD-based method to infer the degree of shared ancestry between polyclonal infections42, implemented in R package dcifer, version 1.2.0 (https://cran.r-project.org/web/packages/dcifer/index.html). Dcifer allows for unphased multiallelic data such as microhaplotypes, explicitly accounts for MOI and population allele frequencies, and does not require densely distributed or linked markers. Additionally, Dcifer provides likelihood-ratio P values adjusted for one-sided tests. Briefly, from the samples, naïve MOI estimates and subsequent estimates of population allele frequencies adjusted for MOI were calculated. We used a likelihood-ratio test statistic to test the null hypothesis that two samples are unrelated (H0: IBD = 0) at significance level α = 0.05 (with the procedure adjusted for a one-sided test). We have calculated the statistical power and false-positive rate (FPR) of our 28 microhaplotype panel at different relatedness levels using two simulation schemes described elsewhere42: (a) no genotyping errors; true MOI and population allele frequencies used as inputs to dcifer, and (b) including genotyping error with fixed error model parameters; MOIs and allele frequencies estimated from these data. The FPR was below the nominal significance level α = 0.05  in a pair of infections with MOI of 2 was 0.91, and 0.72 in a pair of infections with MOI of 3; in our study MOI between most infection pairs was rarely above 2–3. We evaluated the mean IBD and the proportion of related samples within and between districts, ward-level administrative units (shehia) and RACD clusters, and performed one-sided permutation test  to determine which combinations had higher mean IBD and a higher proportion of related samples than expected by chance. Sample pairs were also categorized into groups based on geographic distance into 10 km bins, with an additional category at distance 0 km to capture within-household comparisons. Mean IBD was calculated for each group. We calculated the odds ratios (OR) for finding significantly related infections between or within, shehia, clusters of households covered during an RACD investigation, and individual households. Comparisons of proportions of highly related infection pairs (IBD ≥ 0.9) within and between different groups were assessed using Pearson’s Chi-square test. The igraph package in R75 was used to construct highly related components (referred to as clusters in the results), and to visualize sample pairs at different IBD thresholds to explore them in terms of their spatial and temporal distribution.We estimated pairwise genetic relatedness between infections from the 28 microhaplotypes using WS > 0.95) from the MalariaGEN Plasmodium falciparum Community Project76 to extract microhaplotype data using fasterq-dump (v.2.10.8). Specifically, we used isolates from other sites in Tanzania and Kenya and other regions, including East and West Africa, Southeast Asia, and South Asia , ‘filter’ (%QUAL < 99) and ‘consensus’ functions, and bedtools ‘getfasta’ were used to extract final microhaplotypes , pfdhps (PF3D7_0810800), pfmdr1 (PF3D7_0523000), pfmdr2 (PF3D7_1447900), and pfk13 (PF3D7_1347700). Mutations in pfdhfr and pfdhps mediate resistance to the antifolate drugs sulfadoxine and pyrimethamine80. The pfmdr1 N86 wildtype and 184 F mutant alleles are associated with decreased sensitivity to lumefantrine60, while pfmdr1 86Y mutation and Y184 wildtype alleles confer resistance to amodiaquine (AQ)61. Polymorphisms at positions T484I and I492V in the pfmdr2 gene have been suggested to be involved in artemisinin partial resistance in Southeast Asia63. Mutations in pfk13, including R539T and I543T, are strongly associated with artemisinin partial resistance, resulting in delayed parasite clearance63. Though associated with AQ resistance61, pfcrt was not typed as no amplification product could be obtained with our assay.We measured the prevalence of drug resistant parasites in Zanzibar, focusing on five markers that have previously been associated with resistance to antimalarial drugs: UpSetR package in R81 was used to visualize different combinations of multidrug-resistant parasites. This analysis was limited to monoclonal samples only with complete allele calls at all loci, because haplotype phasing in complex infection was not possible.The mutation frequencies in the population were calculated as the number of samples that contained the mutation over the total number of samples successfully genotyped at each locus. Samples that contained the wild type allele and the mutant allele were classified as “mixed”. Comparisons of prevalence of mutant alleles across districts were performed using Pearson’s Chi-square test. The Further information on research design is available in the Supplemetary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic  stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia ( They are known to have canonical roles in cell proliferation, differentiation, and apoptosis and are characterized by the presence of a leucine zipper (LZ) adjacent to the bHLH domain3. The bHLH and LZ domains are common functional motifs for DNA binding and dimerization that are often also found in other TF families, which indicates that these domains arose from an evolutionarily ancient ancestral TF. MYC TF also harbors a less common motif, a transcriptional regulatory domain, that is less evolutionarily conserved across species than the DNA-binding and dimerization domains4. This transcriptional regulatory domain was shown to strongly control transformation of rat embryo fibroblasts, as evidenced by using a domain deletion mutant5. Moreover, it has been suggested that deletion of any of the conserved domains outlined above can significantly impair MYC function6. Several lines of indirect evidence suggest that MYC TF is involved in governing cellular osmoregulation7. For example, a recent study has revealed that the myo-inositol biosynthesis (MIB) pathway of euryhaline turbot  is positively regulated by MYC, which was demonstrated using an RNAi-mediated knockdown approach8. MYC has also been reported to directly modulate responses to abiotic stressors, including salinity stress, in plants . This modulation is mediated via key hormonal signaling pathways important for plant salinity tolerance11. Such salinity stress-induced, non-canonical roles of MYC TF in plants were also indicated by an in-silico prediction study using bread wheat (Triticum aestivum)12. Moreover, microarray analyses of MYC ko rat cell lines revealed inositol monophosphatase , a key enzyme in the myo-inositol biosynthesis (MIB) compatible osmolyte pathway, as a target gene of MYC13. These studies suggest that MYC TF is important for controlling osmoregulatory mechanisms in eukaryotes.MYC family genes encode class III basic helix-turn-helix (bHLH) transcription factors (MYC TF) that have been evolutionarily conserved in many animal species for at least 400 million years16. This innovative system was initially adopted from bacteria and archaea, in which it had evolved as a pathogen nucleic acid-targeting defense mechanism that conferred resistance to viral infection18. The simplicity and high efficiency of the CRISPR/Cas9 system renders it convenient, cost-effective, and multimodal tool for gene editing in a variety of organisms19. Most studies using this system to date have focused on model organisms such as human cell lines20 and mouse21 both in vivo and in vitro. They have demonstrated the great power of knockout (ko) models for functional studies aimed at causally linking genotypes and phenotypes24. In contrast to the well-established mammalian models, CRISPR/Cas9 approaches have been used to a lesser extent with lower vertebrates such as fish, even though numerous studies have shown that this gene targeting system can be successfully utilized to genetically modify aquaculture fish species in vivo28. For instance, double-allelic ko mutations were introduced in Atlantic salmon  to alter pigmentation25, Insulin-like Growth Factor Binding Protein-2b (IGF-BP2b) was targeted for ko in rainbow trout (Oncorhynchus mykiss)26, alligator cathelicidin gene was targeted in a non-coding region of channel catfish  genome  and myostatin ko mutations were performed in Channel catfish  to improve growth and disease resistance . Examples for in vivo gene targeting in fish also include zebrafish (Danio rerio) where all-in-one CRISPR/Cas9 components were injected into fertilized one-cell stage embryos to generate ko mutants28.The recent revolution of gene targeting approaches by implementing CRISPR/Cas-based approaches has enabled highly accurate and efficient genome editing that is superior to older gene targeting methods such as TALENs or ZFNs, which require covalent linkage of a specific DNA binding domain to a nucleaseOncorhynchus tshawytscha)29. Chinook salmon cell lines were also used to demonstrate the functionality of a vector-based expression system30, as well as to optimize lentivirus-mediated infection for efficient delivery of recombinant DNA into host cells31. Our lab has recently optimized and established a vector-based CRISPR/Cas9 platform for tilapia cell lines32. This study was the first to enable in vitro gene targeting in euryhaline tilapia cells. Our vector-based in vitro approach differs from the in vivo approach used for whole tilapia, which is based on microinjection of gRNA and Cas9 mRNA or protein into Nile tilapia (Oreochromis niloticus) fertilized eggs33.Compared to a considerable number of CRISPR/Cas9 studies in whole fish in vivo, gene editing of fish cell lines in vitro has been rarely used and remains to be explored as a promising tool for high-throughput and low-cost establishment of causality between cellular phenotypes and genomic loci of interest. The first fish cell line that was genetically modified by CRISPR/Cas9 technology was reported for Chinook salmon  are routinely used for short-term loss-of-function studies, interference arising from expression of wild-type or variable mutant proteins remains a concern37. Therefore, recent efforts have been geared towards generating homozygous clonal ko cell models, to ensure that the protein function is completely disrupted and that the resulting mutant proteins that cause effects, are consistent. Several previous studies have reported successful production of clonal ko cell lines for some fish species. For example, Liu et al. have generated a Japanese medaka (Oryzias latipes) ko cell line using RNP transfection for CRISPR/Cas9 gene targeting, followed by isolating a ko clonal cell line having a 9-nt deletion in the sytl5 gene starting with an initial ko cell pool showing 50% gene editing efficiency38. Furthermore, a modified Chinook salmon (Oncorhynchus tshawytscha) cell line that stably expresses Cas9 protein has been used to generate a monoclonal stat2 ko cell line harboring a 2-nt frame-shift deletion in stat239. Such recent efforts to either generate edited pools  or isolate clonal lines  of CRIPSR/Cas9 gene-edited cells can be expanded beyond canonical model species of fish to enable broad comparative and evolutionary studies41.Recent CRISPR/Cas9 approaches aim to obtain highly precise and consistent ko models that are characterized by very high gene editing efficiency and/or clonality, to exclude potentially confounding factors arising from heterogeneity of ko cellsIn this study, genetically engineered polyclonal and monoclonal tilapia cell lines were generated to facilitate studies of cellular functions of MYC TF, specifically its role in osmoregulation. A strategy utilizing a DNA vector-based CRISPR/Cas9 system followed by serial dilution of mutant cells for efficiently isolating clonal ko cell lines has been applied. We present the first successful report of applying targeted gene-editing in combination with serial dilution of a heterogeneous cell population to generate low genetic variability polyclonal and monoclonal tilapia cell lines to enable future functional analyses for assessment of causal genotype–phenotype links.32 previously generated in our lab was used in this study. The genomic presence and expression of Cas9 transgene was verified by an array of PCRs targeting transgene amplicons using both genomic DNA (gDNA) and complementary DNA (cDNA). Cas9-OmB cell working stock  was thawed and maintained at ambient CO2 and 26 °C in L-15 medium  containing 10% (vol/vol) fetal bovine serum , 1% (vol/vol) Penicillin–Streptomycin . When culture plates reached a confluency of 80–90%, cells were passaged  using a 1:5 splitting ratio. For applying hyperosmotic stress to cells, hyperosmotic (650 mOsmol/kg) was prepared using hypersaline stock solution . This stock solution was made by adding an appropriate amount of sodium chloride (NaCl) to regular isosmotic (310 mOsmol/kg) L-15 medium. The hypersaline stock solution was then diluted with isosmotic medium to obtain hyperosmotic medium of 650 mOsmol/kg. Medium osmolality was always confirmed using a freezing point micro-osmometer (Advanced Instruments).A tilapia Cas9-OmB cell lineO. niloticus reference genome deposited at NCBI was used to derive the coding sequence (CDS) for tilapia myca, the MYC proto-oncogene bHLH transcription factor a (Gene ID: 100689989). A myca CDS region spanning exons 2 and 3 was submitted to CRISPOR43 version 5.01 to design small guide RNAs (sgRNAs) for efficient gene targeting of myca. In addition to using CRISPOR, the tilapia myca gene was also analyzed with the CRISPR Knockout Guide Design tool provided by SYNTHEGO (https://design.synthego.com/#/) to design sgRNAs using different algorithms. CRISPOR and SYNTHEGO tools both support convenient tilapia gRNA design by providing an integrated reference genome of O. niloticus (Ensembl 76-Orenil1.0) for calculating off-target effect of sgRNAs by comparison to the whole genome sequences. This aspect of sgRNA design is critical for optimizing specificity. To further validate the selection of the best possible myca sgRNAs, their potential off-targets effects were also manually evaluated using the NCBI reference genome for O. niloticus. Sequences for sgRNA1, sgRNA2, and sgRNA3 were searched against nucleotide sequences using Blastn limited to highly similar sequences (megablast) and restricted to entries associated with the organism “Oreochromis niloticus” (taxid:8128). No off-target genes were identified that matched any of the three top scoring myca sgRNAs suggested by CRISPOR and SYNTHEGO tools. These top three sgRNAs were cloned into an optimized tilapia sgRNA expression vector, as described previously32. Complementary oligonucleotides (Eurofins Genomics) comprising each sgRNA’s forward and reverse sequences (Table 32 using T4 DNA ligase (Promega). The resulting sgRNA expression vectors for myca sgRNAs 1 – 3 were sequenced  and 6 µL ViaFect reagent (Promega) to initiate the formation of transfection complexes. Stabilized transfection complexes yielded after 15 min incubation were then applied to 80% confluent Cas9-OmB cells (P43) by adding the transfection complex solution evenly without any medium change. After 48 h, all medium was removed for transfected cells and a non-transfected control and replaced with 2 mL of L-15 medium containing selection media containing 500 µg/ml hygromycin B . Transfected wells were maintained in selection medium until one day after all cells were detached from the surface of the non-transfected control well. Half of the wells that were transfected with each myca sgRNA were then used for analyzing myca ko efficiency. This was done as previously described32. Briefly, medium was removed and cells surviving hygromycin B treatment were rinsed with Dulbecco’s phosphate buffered saline . They were then scraped from the surface of the well into fresh 0.5 mL DPBS, transferred to a 1.5 mL microcentrifuge tube, and centrifuged for 5 min at 18,000 g. After removal of supernatant, cell pellets were lysed in 20 µl of 25 mM NaOH by incubation at 95 myca test amplicons for analysis of mutational efficiency.For each well of a six-well cell culture plate, two micrograms of TU6m-gRNAscaffHygroR vector containing either myca sgRNA1, sgRNA2, or sgRNA3, the genetic heterogeneity of mutated, selected cells was serially reduced using a limiting dilution strategy to generate more homogeneous ko cell lines. The protocol for this strategy was adapted from a previous publication34 and public protocols  and modified as follows: Selected ko cells were allowed to recover for 14 days in culture to reach 20–30% confluency. They were then harvested as a single cell suspension, counted with a hemocytometer (Hausser Scientific), and diluted to an average concentration of one cell per well before plating into a 24-well cell culture plate. The wells were visually screened periodically to track cells forming colonies using an inverted microscope  for 14 days. When colonies reached 60–70% confluency, they were harvested and split evenly into two new wells of a six-well plate. One well was used for continuous expansion and the other for genotyping the myca mutation in the corresponding cell population.After selection of hygromycin B-resistant cells containing TU6m-gRNAscaffHygroR vector expressing either myca ko cell line was performed by splitting the most promising cell population from dilution round 1  into a 96-well plate at an average density of one cell per well. Cell density was determined by counting cells in a single cell suspension after harvest with hemocytometer (Hausser Scientific). The cell suspension was then diluted to 5 cells per mL medium. Each well received 100 µL of this cell suspension such that the average seeding density was 0.5 cells/well. Seeding an average of 0.5 cells/well ensures that some wells receive a single cell, while minimizing the likelihood that any well receives more than one cell. Cells were maintained for 14 days to track the wells containing a single clonal cell by regular inspection with a microscope .Another round of serial dilution was performed after genotyping the cell populations resulting from the first round of dilution. Further limiting dilution for isolating a single clonal myca was PCR-amplified using appropriate primers  following manufacturer instructions. The test amplicon spanning the targeted region of Cells were seeded in 6-well plates  containing 2 mL complete L-15 medium. After 72 h, attached and proliferating cells were visualized by phase contrast microscopy using an inverted microscope .myca ko truncated protein was compared to wild-type protein. Geneious 2022.0.1  bioinformatics software was used to predict pre-mature translation termination (early stop codon) resulting fromdeletion of a single nucleotide from wild-type mycain a monoclonal ko mutant , see results). The resulting mutant MYC protein sequence was generated by translating the cDNA sequence and compared to the wild-type MYC protein sequence. Both (mutant and wild-type) MYC sequences were annotated with functional domains using InterPro version 90.0 (EMBL-EBI)44. Moreover, the 3D protein structures were predicted for both (mutant and wild-type) MYC proteins and visualized using AlphaFold45,Mol*3D Viewer46, and RCSB Protein Data Bank47. Combined with the above prediction tools, FunFOLDQA48, a protein ligand binding site residue prediction tool, was also employed to reveal whether any DNA binding capacity is preserved in the truncated mutant MYC protein.To confirm functional ko of the resulting truncated protein the structure of 6. In addition to its canonical cellular functions, MYC (encoded by myca) may contribute to tilapia osmoregulation, as myca mRNA is induced during hyperosmolality and multiple MYC binding sites (E boxes) have been identified in the promoter region of the highly hyperosmotically induced tilapia gene IMPA1.1 . To identify the domains that are important for tilapia MYC function and ensure that all functionally important domains are inactivated by CRISPR/Cas9 gene targeting, the tilapia myca gene (Gene ID: 100689989) was identified in the NCBI reference genome sequence for Oreochromis niloticus (NC_031974.2), along with the corresponding mRNA (XM_005448983.4) and protein (XP_005449040.1) sequences. The 432 amino acid sequence of tilapia MYC TF was then used to annotate functional domains using InterPro version 90.044 (EMBL-EBI). This approach identified the long transcriptional regulatory domain at N-terminus and the short DNA-binding domain, consisting of basic helix-loop-helix (bHLH) and leucine zipper (LZ) motifs, at C-terminus  and sgRNA3 (rk#4)—designed with CRIPSPOR and SYNTHEGO both met this criterion, in addition to having the lowest predicted off-target effects. A third sgRNAs—sgRNA1 (248fw) – had the highest scores in both CRISPOR and SYNTHEGO, but was located in the earlier part of the transcriptional regulatory domain (227 bp downstream of the start codon), which means that the truncated mutant protein produced by this sgRNA still contains two highly conserved sequence blocks of the transcriptional regulatory domain .Because highly conserved clusters of sequences were found in the N-terminal transcriptional regulatory domain of tilapia MYC, we aimed to design sgRNAs in the very beginning of the coding sequence, to avoid retention of any potentially functional domain in a truncated mutant protein resulting from frame-shift mutation. Two sgRNAs—myca gene editing experiment was performed with sgRNA1. In this pilot experiment, the sequence of the target locus in Cas9-OmB cells transfected with TU6m-sgRNA1-expression vector and selected with hygromycin was compared to that of wild-type Cas9-OmB control cells at the same locus immediately after hygromycin selection. The Sanger sequencing chromatograms of wild-type and sgRNA1 mutant myca test amplicons as analyzed by TIDE and ICE tools indicated relatively poor gene editing efficiency of 16.9% (TIDE) and 6% (ICE)  Fig. a.Figure myca ko, and eliminate genetic heterogeneity of isolated mutant cell populations, a limiting dilution strategy was employed in a series of experiments that utilized all three myca sgRNAs. In these experiments, cells were allowed to recover from selection by incubation in complete media for 14 days. Cell recovery restored proliferation rate and provided sufficient cell numbers for applying a strategy of limiting dilution. Dilution of the initial myca ko cell mixture into a 24-well plate at an average density of one cell per well  of any single genotype in any of the cell populations isolated by the first limited dilution series . This result suggested that sgRNA1-colony#3 was the most promising for isolating a monoclonal mutant cell line harboring a myca ko cell pools resulting from the first series of limiting dilution. For example, myca ko cells appeared more adherent to each other .2 = 0.99   Fig. a. The ma45, RCSB Protein Data Bank47, and Mol* 3D Viewer46 were profoundly different between wild-type and mutant MYC  outlined for heterogeneous mutant cell populations above, a conspicuous morphological difference was observed for the Cas9-OmB-ons Fig. .Figure 751. MYC has been studied extensively in the context of cancer biology, because of its overexpression in malignant tumors and activation of many hallmarks of cancer52. However, other functions of MYC that are not directly relevant for cancer biology have not received much attention. Since MYC TF is evolutionarily highly conserved and have many transcriptional targets5, they are likely central regulators of the CSR and other diverse cellular functions that are important for normal non-pathological physiology53.MYC is a well-studied transcription factor having numerous cellular functions such as the regulation of cell growth, cell cycle, cell differentiation, global mRNA translation, and cellular stress response (CSR) in a wide variety of organismsmyca ko cell lines. Previous studies suggest that myca, which is the gene encoding for tilapia MYC, may be involved in governing mechanisms of teleost osmoregulation13. Furthermore, our results show that, unlike myc2, myca is elevated at the mRNA level during hyperosmotic stress in tilapia OmB cells . MYC regulates its downstream target genes by binding to the E-box (CACGTG or CATGTG), a MYC-specific cis-regulatory element (CRE).To enable causal links between MYC function and the CSR in tilapia, in particular during salinity (osmotic) stress, this study generated tilapia IMPA1.1 gene encoding the most osmoresponsive enzyme in tilapia . We had previously shown that hyperosmotic transcriptional induction of IMPA1.1 is at least partly mediated by several osmolality/salinity-responsive element 1 (OSRE1) CREs54. The OSRE1 core consensus sequence resembles that of the mammalian tonicity response element (TonE), which is the binding site for nuclear factor of activated T-cells 5 (NFAT5)55. This similarity suggests that tilapia NFAT5 contributes to the osmotic regulation of the IMPA1.1 gene.Intriguingly, three E-box sequences were found in the proximal promoter region (within 1.1 kb of the transcription start site) of the tilapia IMPA1.1 suggests that combinatorial transcriptional regulation involving NFAT5 and MYC controls the hyperosmotic induction of tilapia IMPA1.1. To enable functional analyses of the role of MYC and its interaction with NFAT5 for osmoregulatory target gene transactivation this study generated multiple poly- and mono-clonal myca ko tilapia cell lines that can be used in combination with reporter assays57, molecular and cellular phenotyping60, and other approaches62 to comprehensively characterize the role of MYC for teleost osmoregulation.However, gene expression is often regulated in a combinatorial manner and depends on multiple different CREs and TFs. Our findings of hyperosmotic MYC TF mRNA elevation and the presence of multiple MYC CREs (E-boxes) in 34. Such highly heterogeneous polyclonal cell pools can have poor overall gene editing efficiency due to inefficient delivery or low sgRNA-dependent mutational efficiency64. Moreover, some of the cells in this heterogeneous pool may harbor non-specific mutations that can cause off-target effects. One way to eliminate such potential off-targets is to isolate genetically distinct cell pools and check for consistent phenotypes when multiple of these distinct pools are used for experiments that evaluate functional consequences of gene ko. In this study, we applied a serial limiting dilution strategy to increase gene editing efficiency and decrease the genetic heterogeneity of polyclonal and monoclonal mutant (gene-edited ko) cells. Both polyclonal and monoclonal cell lines are useful depending on the purpose of the subsequent experiment39.Targeting genes in vitro using a CRIPSR/Cas9 system results in a heterogenous pool of edited cells with varying indel populationsWe successfully produced ten polyclonal cell pools with reduced heterogeneity in the indel genotypes when compared to the original mixture of gene edited cells. Five of these polyclonal lines had much higher gene editing efficiencies (> 80%). The remaining five cell pools with < 80% gene editing efficiency also showed improved mutational efficiency relative to the starting population. The high gene editing efficiencies achieved during the initial series of limiting dilution illustrate that this strategy represents a rapid way for enrichment of desirable genotypes. However, wildtype mRNA and protein expression from unedited or heterozygous cells harboring a haploid mutant genotype represents a potential pitfall of studying polyclonal ko cells. This would be particularly problematic for long-term studies if unedited or haploid cells have a growth rate that exceeds those of diploid mutants.myca gene after only two rounds of limiting serial dilution. In theory, it is possible to decrease the cell concentration during the first round of dilution even further and seed more aliquots  to obtain monoclonal lines after a single limiting dilution step. However, in praxis, our approach of subsequent serial dilutions at an average seeding density of 1 cell per well followed by an average seeding density of 0.5 cells per well yielded the best results with cells retaining their ability to form colonies within a short time (within 4–5 weeks). This approach also reduced the likelihood of seeding cell clusters present in cell suspensions during the second round of limiting dilution35. Thus, limiting dilution is an effective strategy to overcome (1) the genetic heterogeneity of mutant cells, (2) the potential presence of unedited or haploid mutant cells in mixed populations, and (3) the low delivery efficiency of vector-based CRISPR/Cas9 in cultured fish cells67. The limiting dilution strategy is not only fast but also very cost-effective.To eliminate concerns about possible phenotype masking effects due to the potential presence of unedited or haploid genotypes in a polyclonal cell pool, we performed another series of limiting dilution. For this second dilution series, a polyclonal cell line from the first series of dilution was chosen, which had the highest frequency of frameshift  mutations as determined by ICE analysis. We demonstrate that it is possible to isolate a monoclonal tilapia cell line harboring a 1-bp deletion in the 68. For example, workflows for isolating gene-edited clonal cell lines by fluorescent-aided sorting system are currently only feasible for mammalian cells69. Moreover, even if expensive cell sorting devices and corresponding labeling approaches are available, cells would be potentially exposed to the non-sterile conditions and in danger of being contaminated. Cell viability is also negatively affected by the cell sorting process. The serial limiting dilution strategy used in this study provides a robust, rapid, and cost-effective platform for generating ko clonal cell lines for studies of mutagenesis effects on cellular phenotypes of teleost fishes.Our approach overcomes difficulties often encountered when attempting to isolate clonal mutant cell lines from non-canonical model organisms70 has been widely adopted for mutation detection since its development in 2014 as an accurate, versatile, and time-saving alternative to restriction enzyme-based assays71. The ICE analysis pipeline (SYNTHEGO)72 has been developed more recently and examined rigorously by comparing to NGS-based amplicon sequencing data. This evaluation revealed that the accuracy of ICE analysis is comparable to that of NGS-based approaches such as CRISPResso2, which aligns deep sequencing reads to a reference sequence73.To analyze results from CRISPR/Cas9 gene editing experiments, the test amplicon sequence spanning the target site must be analyzed with bioinformatics tools that deconvolute the indel heterogeneity into interpretable scores. We used TIDE and ICE analyses for this purpose. Sanger sequence chromatograms of test amplicons from genomic DNA of gene edited and wildtype cells and the corresponding sgRNA sequence were used as the input data. TIDE74.In addition to scoring the overall gene editing efficiency, ICE also provides a Knockout score (KO-score) which is a useful measure to determine how many of the contributing indels are likely to result in a functional ko of the targeted gene. The main advantages of using both TIDE and ICE in combination are being able to: 1. Compare the consistency of two independent measures for gene editing efficiency, which are derived from different analytical algorithms, 2. Obtain detailed information about distribution and frequency of different types of indels, and 3. Estimate the frequency of indels resulting in a functional ko. Because of these advantages and their complementary features, the combined use of TIDE and ICE for indel heterogeneity analyses has been reported previouslyOne important finding of our study was a substantial difference between KO-score and indel efficiency observed in the sgRNA1-colony#4. While TIDE and ICE indel efficiency scores for this cell population were 98.4% and 92%, respectively, the KO-score was only 42% Fig. . This inThe scores provided by TIDE and ICE analyses provide effective and unbiased selection criteria of choosing a particular population of cells from the first round of limiting dilution for subsequent serial dilution of cells. We selected the sgRNA1-colony#3 cell population resulting from the first limiting dilution for the second round of limiting dilution because of the high abundance of a specific genotype (a 1-bp deletion indel accounts for 51.4%) and because the frameshift mutation introduced a premature translation termination codon shortly after the target site, Fig. 75, medaka76, and Chinook salmon38. These studies were focused on investigating mechanisms of resistance to viral infection. However, generation of polyclonal or monoclonal ko cell lines from heterogeneous indel genotypes generated by CRISPR/Cas9 technology, whether by limiting dilution or other approaches, has not been reported prior to this study for any tilapia species.The limiting dilution strategy had already been successfully used in a few cases to generate clonal ko cells in different fish species including carp78. Tilapia are widely used aquaculture species, second only to carp regarding global production yields. However, like many other organisms, tilapia are subject to climate change and pollution, which negatively affects their performance and natural habitat and aquaculture. Mechanistic insight derived from gene targeting studies helps to understand, properly interpret, and compensate for such impacts to facilitate mitigating these negative effects.Here we established proof of principle that monoclonal tilapia ko cells can be generated by limiting dilution of heterogeneous indel genotypes resulting from using a vector based CRISPR/Cas9 system with cell lines. Such monoclonal ko lines have a defined genotype that facilitates the interpretation of functional studies aimed at evaluating the effects of gene inactivation on cellular phenotypes, for example osmoregulatory, disease resistance, proliferation, or other phenotypes that are informative for understanding basic physiological mechanisms but also of great interest from an applied perspective, e.g. for improving aquaculturemyca gene encoding MYC TF was chosen as to demonstrate the effectiveness of the limiting dilution strategy for generating a clonal ko cell line. This cell line and the polyclonal myca ko lines generated in the present study enable testing the role that MYC TF plays for tilapia cellular osmoregulation, its contribution to the activation of the myo-inositol biosynthesis (MIB) pathway, and its contribution to other cell functions. This approach can be extended to other targets and species of interest, for example genes important for aquaculture traits other than salinity tolerance and candidate genes other than myca, including those identified by previous GWAS and SNP analyses in multiple aquaculture fish species81. These ko cell lines allow for deep functional analyses that associate specific genotypes with complex phenotypes, including systems level molecular phenotypes revealed by transcriptomics82 and proteomics83.In the current study, the myca clonal ko cell line did not differ from that of wild-type Cas9-OmB cells. This contrasts with several polyclonal ko cell pools obtained after the first round of limiting dilution, which displayed reduced proliferation even after three passages. This result indicates that myca ko is not lethal, and even does not hamper cell propagation in vitro. This phenomenon is interesting as we expected that all ko cell lines might show similar reduced cell viability and slower cell growth compared to the wild-type cells because of the known essential roles of MYC in numerous cellular processes including cell growth, proliferation, and differentiation84. Nevertheless, the aberrant morphologies of tilapia myca ko cell lines (Figs. Surprisingly, the cell viability and growth rate of the mycako cells in this study, more in-depth molecular phenotyping will help understand the physiological consequences of mycako in future studies. Specifically, osmotolerance phenotypes of myca ko cells can be analyzed42 and quantitative proteomics approaches can be employed to provide functional insights into biochemical and genetic networks that are controlled by MYC TF86. The limiting dilution approach and the myca ko tilapia cell lines generated in this study will empower such future functional analyses.Although aberrant cell morphology is reported for myca ko cell lines for in depth cellular phenotyping studies, directed at investigating functions of MYC TF in tilapia.In summary, this study has successfully used a vector-based CRISPR/Cas9 approach in combination with a serial limiting dilution strategy to generate mono- and poly-clonal tilapia Supplementary Information.


Such assembly can be performed at temperaturesas high as 550 °C. This large temperature range significantlywidens the portfolio of nanoparticle fabrication techniques, allowingus to demonstrate a variety of chiral nanoparticle assemblies, rangingfrom metals , semiconductors (Ge), and compound semiconductors(GaAs) to oxides (WO3).Fabrication of chiralassemblies of plasmonic nanoparticles isa highly attractive and challenging task, with promising applicationsin light emission, detection, and sensing. So far, primarily organicchiral templates have been used for chirality inscription. Despiterecent progress in using chiral ionic liquids in synthesis, the useof organic templates significantly limits the variety of nanoparticlepreparation techniques. Here, we demonstrate the utilization of seeminglyachiral inorganic nanotubes as templates for the chiral assembly ofnanoparticles. We show that both metallic and dielectric nanoparticlescan be attached to scroll-like chiral edges propagating on the surfacesof WS The large discrepancybetween the size of the molecules and the wavelength of light, however,severely hinders these promising applications. It is therefore desirableto prepare chiral nanostructures and metamaterials that exhibit exceptionallystrong light–matter interactions, potentially increasing thesensitivity and throughput wherever the chirality of light–matterinteractions comes into question.Many important biomoleculesand pharmaceuticals are chiral because of the inherent chirality oflife on our planet.g-factor), defined as the ratioof differential extinction of circularly polarized light to totalextinction. Very large g-factors can be reached bysqueezing the light waves down to the length scale of biomoleculesby carefully designed plasmonic3 or dielectric5 nanostructures. The synthesis approaches in this context rely onusing chiral seeds or chiral ligands, and thus, chirality can be inscribedinto nanoparticles even when achiral building blocks are used7 and at different length scales.8 Theirchiroptical response can be further enhanced by inscribing a long-rangechiral order to extended clusters of nanoparticles.11 Such rationallydesigned assemblies can be tailored to exhibit g-factorsin the visible part of the electromagnetic spectrum, enhanced by higher-ordercollective interactions.13 These chiral superstructuresare an attractive candidate for plasmon-enhanced chiral sensing wherethe single-molecule sensing potential of plasmonics14 is utilized in the realm of chirality analysis.15 Although there is an ongoing debate whetherachiral sensing platforms could be superior to the chiral ones,16 the sensing capability of chiral superstructureshas already been demonstrated in the detection of markers of Parkinson’sdisease17 or in the detection of attomolarDNA concentrations.18 Going beyond sensing,chiral nanoparticle assemblies can be also used for driving chiralphotochemistry and facilitating enantioselective chemical synthesis,20 which could have a disrupting effect on production of pharmaceuticals.Chiral nanoparticle assemblies could also serve as sources or detectorsof circularly polarized light,22 of dynamic chiral nanomachines,23 or in chiral phototherapy.24The strength of chiropticaleffects can be characterized by thedissymmetry factor  and evendisorder of the original helical arrangement.40The strategies used to prepare chiral nanoparticleassemblies comprisetwo approaches: top-down and bottom-up. The former, usually lithography-based,yield complex geometries42 or perovskite nanocrystalassemblies.43 Moreover, these studies demonstratedonly a wet chemistry-based approach, which is limited to in-pot chemicalreactions and their products, similar to “soft” templates.Going beyond this approach, here we utilize chiral grain boundariespresent on the surface of inorganic nanotubes made of a layered vander Waals material (WS2) that serve not only as attachmentand nucleation sites for nanoparticles prepared by wet chemistry butalso during evaporation under vacuum and high-temperature conditions.The resilience of inorganic nanotubes with respect to high temperaturesopens the possibility of bottom-up fabrication of chiral assembliesof nanoparticles made of a much broader group of materials, includingthose not available so far, like germanium, gallium, or tungsten trioxide.Utilizing a “hard” inorganic template wouldnaturallyoffer more degrees of freedom for its further processing steps orfunctionalization with optically active nanoparticles. However, suchsynthesis has been demonstrated only using silica nanohelices so far,providing a template for gold45 and are thus more vulnerable to chemicalattacks such as oxidation or reduction. For example, copper oxidebegins to form on a graphene-covered copper foil just beneath thegrain boundaries of the polycrystalline graphene, serving as a toolto visualize these otherwise hardly visible boundaries46 (see 247 (see 49 the line defects (unsaturated sulfur bonds in the case of WS2) are then the only chemically active sites.51 Due to the tubular shape of the surface, a single-line defect couldin principle form a chemically reactive three-dimensional spiral , which allows for selective attachmentof various molecules or nanoparticles into a helical assembly. Toverify this hypothesis, we decorated a large batch of WS2 nanotubes with gold nanoparticles using an established one-pot chemistryprotocol (see the Line defects (grain boundaries or step edges) on the surfaces ofsolid-state materials exhibit higher reactivity than the defect-freeareases46 see 1a. Notab 247 see 1b. As th see the 1c.2 formation in a growth reactor. Both scenarios describe the sameprocess: sulfurization of tungsten oxide nanowires in H2S gas during the formation of WS2 nanotubes in a growthreactor.55 We utilize an analytical model of domain spreading in a tubulargeometry . Forsimplicity, we have assumed that growth of an initial WS2 domain on the surface of WOx nanowire could occur from only a singlenucleation site . We assumethis initial domain to be of triangular shape . This line defect is characterizedby a chiral angle, β . We will show later that this scenariodoes not predict all of the experimental results. Nevertheless, itcould potentially reproduce line defect formation under differentnanotube growth conditions or in different material systems. The secondscenario corresponds to overgrowth of an already formed nanotube byan additional WS2 layer, e.g., from a supersaturated vaporduring the growth reactor cooling down. 2 in particular.57 In contrast to the firstscenario, the propagating layer was allowed to climb over the otheredge upon collision and continued to grow laterally as an overlayer. Additionally, a transmission electronmicroscopy (TEM) analysis of the nanotubes decorated with nanoparticlehelices confirmed the scroll-like nature of their outer walls  and were always able tofind a match between the chiral angles measured from the respectivediffraction pattern and those measured directly from an SEM imageof the nanoparticle chains, thus generalizing our conclusions. A thoroughTEM analysis allows us to go even beyond just chiral angle determination:it is possible to identify the nature of step edges . We have identified them to be of zigzag type, which is consistentwith predictive models using density functional theory.56 Having identified the chiral lines as step edgesinstead of grain boundaries makes it possible to utilize the stepedge as a nucleation site for adatoms diffusing along the nanotubesurface, e.g., during vapor deposition. We demonstrate this capabilityin the last part of the paper.g points 1d. Figurg points 2b is alwg points 2a. We ha42 However, the advantage of our inorganic tubular templates over “soft”organic templates is the possibility of utilizing higher process temperatures;therefore, a wider variety of materials for nanoparticle assembliescan be used. This is demonstrated in 2 nanotube has beencontrollably oxidized in water vapor at 300 °C,47 leading to the formation of WO3 nanoparticlesalong the edge of the outer wall. The very high temperature stabilityof WS2 nanotubes 47 also offers flexibility in terms of deposition techniques. 2 nanotubes decorated with gallium and germanium nanoparticles,which were deposited by evaporation of the respective elemental materialsunder high-vacuum conditions onto nanotubes dispersed over a solidsubstrate (silicon). Note that gallium could be subsequently oxidizedto form gallium oxide, nitrided to form gallium nitride,58 or exposed to group V atomic flux to form III–Vsemiconductor ,thus widening the portfolio of available nanoparticles. There areno limitations other than the deposition temperature that would preventother techniques like atomic layer deposition59 or chemical vapor deposition60 from beingused for creation of chiral nanoparticle assemblies utilizing theseinorganic templates.Gold nanoparticle assembliessimilar to those studied above havebeen previously demonstrated .2 nanotubesdiscussed hereare rarely found within bare unsorted nanotube batches because thesynthesis processes for multiwall WS2 nanotubes with smoothouter surface are currently well developed.54 Here, we have shown that the initially undesired scroll-like productsof nanotube synthesis can become templates for further processingtoward very attractive nanostructures. Hence, further effort is necessaryto identify the growth conditions resulting in the formation of scroll-likenanotubes.. We hypothesize that the scroll-like overlayer forms whena reactor slowly cools from growth temperature during the preparationof WS2 nanotube templates. At lower temperatures, it isplausible that the vaporized transient tungsten oxides resulting fromthe nanotube formation process condense on the outer walls of a fewnanotubes and react with the H2S residues in the reactor.This CVD-like reaction results in WS2 nucleation and overlaygrowth on the surface of existing WS2 nanotubes followingthe scenario depicted in Figure S10).What seems to be difficult to control is the particular handedness.The nanostructures discussed here are inherently racemic; to extractonly single handedness, a specific postfabrication sorting techniquehas to be used. Nevertheless, tubular inorganic templates must notbe limited only to nanotubes: nanoscrolls formed by rolling up 2Dmaterials have been experimentally observed since 2003.63 For example, carbon nanoscrolls can withstand even higher temperaturesthan TMDs and thus could serve as templates for formation of nanoparticlechains that require even higher synthesis temperatures than shownin this study. Still, despite recent attempts to clarify the rollingprocess of planar 2D layers, the overall understanding of the formationof the nanoscrolls is still poor.65 With furtheradvancements in the preparation of these nanostructures, the utilizationof the  defect lines for nanoparticle attachment representsa promising way toward  nanoparticle assemblies of variousmaterials and geometries.Note that the scroll-like WS2 nanotubes are stable up to 550 °C ina vacuum. Taking advantage of the very high temperature stabilityof these templates, we have demonstrated the possibility of preparinga wide range of nanoparticle assemblies .Using physical vapor deposition allowed going beyond in-pot chemicalsynthesis which has been exclusively utilized so far in connectionwith organic chiral templates.66 Additionally, scroll-like nanotube templates permit to inscribechirality to otherwise challenging material systems,67 like the tungsten trioxide demonstrated here. The versatilityof our approach holds promise for the fabrication of chiropticallyactive building blocks in emerging areas of chiral nanophotonics,where facile fabrication of such assemblies represents a great challenge.The inherent crosstalk between circular and linear dichroisms69  preventedus to confirm the characteristic chiral signatures present in opticalspectra. With more advance techniques,70 such analysis should be possible and could, for example, improveunderstanding of semiconductor chiroptical nanomaterials that remainselusive, despite their attractiveness in many fields.71 Besides, TMD templates covered with plasmonic nanoparticlescould become an ideal playground for studying chiral plasmon–excitonpolariton complexes.72 The proposed fabricationapproach thus significantly advances the so-far narrow range of preparationtechniques of chiral nanomaterials, allowing for experimental studiesof complex chiral systems that have been unavailable up to now.In summary, we have demonstrated thefabrication of metallic, semiconductor,and oxide nanoparticle chiral assemblies by utilizing scroll-likeinorganic nanotube templates. In contrast to the commonly used organictemplates, WS2 nanotubes were prepared via “vapor–gas–solid(VGS)” method, as discussed in detail in our previous reports.55 In brief, the WO3 precursor was initially reduced totungsten suboxide (WO2.75) phase using H2 gasas a reducing agent, which results in the formation of 1D WO2.72 suboxide nanowhiskers. The as-formed WO2.72 nanowhiskersundergo further sulfurization as a “solid–gas”reaction under the continuous flows of H2S and H2 gases to convert suboxides into multiwalled, hollow tungsten disulfidenanotubes.TheWS2 nanotubes with gold nanoparticles using achemical procedure described in ref  using a combinationof an ultrasonic bath and subsequent mechanical shaking; each stepwas repeated at least four times. The WS2 nanotube dispersionwas then added to a hot-plate-heated beaker with a boiling 0.043 mMaqueous solution of HAuCl4 under vigorous stirring. Thedispersion was still being heated to boil for an additional 3 min,and subsequently the hot plate was turned off and allowed to coolslowly to room temperature. This procedure resulted in WS2 nanotubes decorated with gold nanoparticles with an average diameteraround 10 nm. By varying the respective ratio between WS2 and HAuCl4, e.g., by changing the HAuCl4 concentration,the average diameter of the AuNP could be tuned between 5 and 30 nm.The resulting dispersion was then drop-casted onto a cleaned substrate.Wedecorated WSd in ref . Brieflyp = 1 × 10–10 mbar) equipped withgallium, germanium, and arsenic evaporators. The sample was firstannealed using a calibrated pBN heating element at 350 °C for20 min to degas any IPA residues and then stabilized at growth temperature. Thegrowth temperature, flux, and overall duration were optimized to achievenanoparticle formation on the chiral line only. For gallium, the optimumflux was 1.08 nm/min for 2 min. For germanium, the optimum flux was0.05 nm/min for 10 min. In the case of droplet epitaxy demonstratedin Figure S9e, the WS2 nanotubeswith Ga nanoparticles were exposed to As flux  at elevated temperature (215 °C) foran additional 1 h.As described above, the nanotubeswere dispersed in IPA using anultrasonic bath and mechanical shaking. Next, the solution was drop-castedonto a silicon substrate and allowed to dry. The resulting samplewas inserted into a complex of ultrahigh-vacuum chambers  or ex situ by a Kratos Axis Supra. The peak fitting details as well as XPSacquisition conditions are described in the Supporting Information.For EDXanalysis, the NTs(either with in-pot-prepared Au NPs or just bare) were dispersed ontoa standard 300-mesh Mo grid. In the latter case, the dispersion wasfollowed by evaporation of specific chemical element under vacuumor by oxidation to get WO


Optical tweezers have profound importance across fields ranging from manufacturing to biotechnology. However, the requirement of refractive index contrast and high laser power results in potential photon and thermal damage to the trapped objects, such as nanoparticles and biological cells. Optothermal tweezers have been developed to trap particles and biological cells via opto-thermophoresis with much lower laser powers. However, the intense laser heating and stringent requirement of the solution environment prevent their use for general biological applications. Here, we propose hypothermal opto-thermophoretic tweezers (HOTTs) to achieve low-power trapping of diverse colloids and biological cells in their native fluids. HOTTs exploit an environmental cooling strategy to simultaneously enhance the thermophoretic trapping force at sub-ambient temperatures and suppress the thermal damage to target objects. We further apply HOTTs to demonstrate the three-dimensional manipulation of functional plasmonic vesicles for controlled cargo delivery. With their noninvasiveness and versatile capabilities, HOTTs present a promising tool for fundamental studies and practical applications in materials science and biotechnology. Traditional optical tweezers require high laser powers risking photothermal damage of the trapped objects. Here, the authors present hypothermal opto-thermophoretic tweezers (HOTTs), which use environmental cooling to simultaneously enhance thermophoretic trapping force at low laser powers and suppress the thermal damage to trapped objects. Optical tweezers trap target objects by the gradient force, which depends on the refractive index, particle size, and laser wavelength5. High laser power is usually required to trap nanomaterials and biological objects that have a low refractive index contrast with their surroundings, which can induce damage to the materials and reduce the cell viability6.The development of optical tweezers has led to tremendous advances in many fields such as optical nanomanufacturing10, opto-electronic tweezers12, and opto-acoustic tweezers13, have been developed. However, they are usually limited by specific substrates, complex setups, or confined working ranges. Recently, optothermal tweezers have been developed to achieve versatile manipulation of colloidal particles under a light-controlled temperature gradient18. While optothermal tweezers enable enhanced trapping capability with a laser power that is 2–3 orders of magnitude lower than optical tweezers19, optical heating can cause thermal stress and degradation to the particles and biological cells. In addition, since many colloids and cells show a thermophobic behavior and move away from the laser heating spot, optothermal tweezers require additional surfactants or salts to tune their thermophoretic response23. However, trapping cells and biological objects in required fluidic environments is often essential for biological applications to reduce the effect of additives and elucidate the bio-physio-chemical interactions of the cells26. Without any solution modification, specialized traps such as thermal Paul trap and anti-Brownian electrokinetic trap were implemented to trap thermophobic objects with limited accuracy and manipulation abilities29.To overcome these challenges, different variations of optical tweezers, such as plasmonic tweezersIn this work, we propose hypothermal opto-thermophoretic tweezers (HOTTs) to overcome these limitations. Specifically, we couple environmental cooling and localized laser heating to achieve low power thermophoretic trapping of target objects and simultaneously avoid optical and thermal damage. More importantly, this cooling strategy also plays a vital role in facilitating the thermophilic behavior to enable the trapping of diverse colloids at different conditions. We also demonstrate the successful trapping and manipulation of fragile erythrocytes in different tonicities to resemble different bio-physio-chemical functionalities. We further show the capability of HOTTs for three-dimensional manipulation (3D) of plasmonic vesicles for light-controlled drug delivery.30, which can be expressed as32. Mainly, temperature plays a significant role in affecting the particle-solvent interactions. The aqueous solvent used in several biological applications exhibits thermo-polarization of water molecules, creating a thermoelectric field that inverts as the temperature changes. This is mainly due to the strong dependence of the quadrupolar component of the thermoelectric field on the temperature that is determined by the thermal expansion of water33. The resultant 3435. At the ambient temperature, 0C, the PS microparticle was repelled away from the laser beam due to a net repulsive thermophoretic force , and solutions (DI water and electrolytes). In all the conditions, the trapping stiffness increases by 3–5 times with the reduced environmental temperature, showing the versatility of HOTTs. Moreover, by varying the composition of the electrolyte (‘x’ in NaClxOH1-x), we demonstrate that trapping ability is induced in a wider range of compositions at reduced environmental temperature , the thermoelectric response is additionally dependent on the surrounding particle’s position distribution, which continuously varies due to the Brownian motion of the particle, thus requiring statistical analysis to determine the thermoelectric response of single particles. Accordingly, we focus the laser beam on single particles to determine the instantaneous trapping probability, which fluctuates between 10–40% at the ambient temperature at concentrations from Φ > 0.01,  serve as potential markers for pathophysiological disease diagnostics like sickle cell anemia46 and malaria47. The tonicity of extracellular fluid alters the shape and size of erythrocytes, which has been recently used to determine the severity of illness caused in SARS-COV-2 patients48. Although optical trapping of erythrocytes in varying tonicities can enable the extension of these cell-cell interactions and biomedical studies for diverse diseases, it is challenging because of the erythrocyte’s non-linear optical response50 and photo-damage51.Erythrocytes  are important biological entities that are currently used in drug deliveryHere, we demonstrate the capability of HOTTs for biological applications by trapping erythrocytes in different tonicities at low laser power, while retaining their structural integrity. The tonicity of the extracellular fluid is altered by tuning the concentration of phosphate buffered saline (PBS) solution , the salt concentration in the extracellular fluid is lower than the erythrocyte. The osmotic gradient across the membrane results in the permeation of water into the cell and the swelling of the cell to become nearly spherical Fig. . The celWhile their structural integrity remains intact, further assay experiments are necessary to verify the preservation of cellular physiological conditions following laser exposure. Decreasing the duration of laser exposure would aid in maintaining the physiological conditions of the cells. Incorporating alternative engineering methods that employ focused laser beams to attract cells while gradually increasing the focal beam waist as cell approaches the laser beam (thus reducing the temperature gradient) would additionally contribute to the preservation of cellular integrity.55. Plasmonic vesicles are gold-coated vesicles with controlled optical and spectroscopic properties for diverse biomedical applications59. Although optical and optothermal trapping of naked vesicles or gold nanoparticles has been achieved61, optical trapping of plasmonic vesicles is challenging due to the large plasmon-enhanced scattering force because of the gold layer. Also, the heat produced during laser illumination causes an uncontrollable thermophoretic force which usually directs the vesicle towards the cold (away from the laser beam) at ambient temperature62. In addition, the heat generated also induces drug release from plasmonic vesicles. The capability of trapping while maintaining the integrity of plasmonic vesicles will enable precise positioning followed by optically triggered drug release, and it holds great promises in several applications.Extracellular and synthetic vesicles have shown great importance in bioimaging, drug delivery, biological transport processes, and therapeutics63. Meanwhile, the high optical scattering force causes the vesicle to repel away from the focus of the laser beam at the ambient temperature irrespective of the thermophoretic force direction  by suppressing thermal damage. This capability is desired for a variety of biological applications, including drug development and cell-cell interactions. Although we successfully demonstrate the trapping of red blood cells in diverse tonicities while retaining the structural integrity, additional studies are essential to successfully characterize the metabolic activity of the trapped cells after laser exposure. We further exploited HOTTs for 3D manipulation of thermosensitive plasmonic vesicles and demonstrated the light-controlled cargo release. HOTTs can additionally be extended to non-plasmonic targets by using a plasmonic or a light-absorbing particle as a delivery agent2 particles are purchased from Bangs Laboratories. COOH- functional PS particles of 2.67 μm particles are purchased from Bangs Laboratories. Erythrocytes are purchased from Human Cells Bio and stored at 4 °C. All experiments involving erythrocytes are performed within 4 weeks of the extraction date, and new erythrocyte samples were prepared for every experiment.PS microparticle suspensions of 0.96 μm and 2 μm are purchased from Thermo Fisher Scientific. 0.5 μm PS particles and 1.96 μm SiO−7 torr at a rate of 0.1 nm/s. Later, gold-deposited coverslips are thermally annealed at 550 °C for 2 h . For microparticle experiments, thermally annealed substrates are used as prepared after cleaning using DI water and nitrogen gun. For erythrocyte experiments, the substrates are immersed in a 1 mM 11-mercaptohexanoic acid in ethanol to prevent the adhesion of red blood cells onto the substrate. The modified substrates are cleaned using water droplets (gentle cleansing) to remove excess solution for uniform functional layer formation.Glass coverslips were triple-rinsed with iso-propyl alcohol and water and cleaned under a nitrogen gun. The coverslips were then loaded into a thermal evaporator (Kurt J Lesker Nano36), and 4.5 nm gold films are thermally deposited at a pressure of 1 × 1064: first, Dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a 4:1 molar ratio were dispersed in chloroform and dried with N2, followed by overnight evaporation under a vacuum. The dry lipid film was then dispersed in 10 mM PBS containing calcein 75 mM for 1 h and subsequently extruded through 400 nm polycarbonate membranes for 11 passes using Avanti Mini Extruder (Avanti Polar Lipids). Free calcein was removed by centrifugation at 5000 g for 10 min and then washed with PBS three times. Second, gold nanoparticles were decorated onto DPPC liposome using the in-situ gold reduction method. Aqueous solutions of gold chloride (10 mM) and ascorbic acid (40 mM) were prepared. Gold chloride solution was added and gently mixed with liposome suspension (1.5 mM lipid concentration) in a molar ratio of 1:4 until uniformly distributed, followed by the addition of the same volume of ascorbic acid solution. Following reduction, plasmonic vesicles were separated from unreacted ascorbic acid and gold chloride by centrifugation  and then stored at 4 °C until use. For trapping experiments, plasmonic vesicles that are in the sub-micrometer regime (800–1500 nm) are used due to their easy visualization under the microscope.Plasmonic vesicles were prepared via a two-step method following a previously reported method with minor modifications as followsFor substrate-based trapping, a 532 nm laser beam (Laser Quantum Ventus 532) is passed through a 5X beam expander and directed into the objective  of an optical microscope (Nikon Ti-E) through a series of reflective mirrors. The liquid sample containing the target objects are loaded into a 120 μm thick spacer that acts as a microchamber. A charged coupled device (CCD – Nikon DS-Fi3) is used to visualize and record particle-trapping videos. Phase camera  is used to evaluate the temperature increase because of laser heating of the substrate. The sample is placed on an aluminum sample holder that provides edge-support on all sides of the coverslip. The Peltier thermoelectric cooler  along with the aluminum heat sink is then rested on top of the glass coverslips. The weight of the heat sink ensures perfect contact of the thermoelectric cooler with the sample and the temperature resistance across the interface is assumed to be negligible. An annular cooler is selected to enable the light path through the device. A corresponding through-hole is drilled into the heat sink, for white light to travel through the sample and reach the camera.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Reporting SummarySource Data