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The mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system

Heredity volume 134, pages 221–233 (2025)Cite this article

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Abstract

The effective use of CRISPR technologies in emerging model organisms faces significant challenges in efficiently generating heritable mutations and in understanding the genomic consequences of induced DNA damages and the inheritance patterns of induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for gene editing in the microcrustacean Daphnia pulex; 2) assessing the editing dynamics of Cas9 and Cas12a nucleases in the scarlet knock-out mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet gene. Our reengineered microinjection method results in efficient biallelic editing with both nucleases. Our data suggest site-specific DNA cleavage mostly occurs in a stepwise fashion. Indels dominate the induced mutations. A few, unexpected on-site large deletions (>1 kb) are also observed. Notably, genome-wide analyses reveal no off-target mutations. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progeny. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes involved in human neurodegenerative diseases.

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Fig. 1
Fig. 2: The microinjection workflow, guideRNA locations, and scarlet knockout mutant phenotype.
Fig. 3: Examples of transmission pattern of scarlet knock-out genotype.
Fig. 4: Examples of Cas9/Cas12 induced mutations.
Fig. 5
Fig. 6

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Data availability

DNA and RNA sequence data are available at NCBI Sequence Read Archive under project ID PRJNA1055485 and PRJNA1060702, respectively.

References

  • Adikusuma F, Piltz S, Corbett MA, Turvey M, McColl SR, Helbig KJ, Beard MR, Hughes J, Pomerantz RT, Thomas PQ (2018) Large deletions induced by Cas9 cleavage. Nature 560:E8–E9

    Article  CAS  PubMed  Google Scholar 

  • Aird EJ, Lovendahl KN, St Martin A, Harris RS, Gordon WR (2018) Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol 1:54

    Article  PubMed  PubMed Central  Google Scholar 

  • Akcimen F, Lopez ER, Landers JE, Nath A, Chio A, Chia R, Traynor BJ (2023) Amyotrophic lateral sclerosis: translating genetic discoveries into therapies. Nat Rev Genet 24:642–658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alarcon M, Abrahams BS, Stone JL, Duvall JA, Perederiy JV, Bomar JM, Sebat J, Wigler M, Martin CL, Ledbetter DH et al. (2008) Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Am J Hum Genet 82:150–159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alexa A, Rahnenfuhrer J (2019) topGO: Enrichment Analysis for Gene Ontology. R Package

  • Altshuler I, Demiri B, Xu S, Constantin A, Yan ND, Cristescu ME (2011) An integrated multi-disciplinary approach for studying multiple stressors in freshwater ecosystems: Daphnia as a model organism. Integr Comp Biol 51:623–633

    Article  CAS  PubMed  Google Scholar 

  • Anaka M, MacDonald CD, Barkova E, Simon K, Rostom R, Godoy RA, Haigh AJ, Meinertzhagen IA, Lloyd V (2008) The white Gene of Drosophila melanogaster Encodes a Protein with a Role in Courtship Behavior. J Neurogenet 22:243–276

    Article  CAS  PubMed  Google Scholar 

  • Aryal NK, Wasylishen AR, Lozano G (2018) CRISPR/Cas9 can mediate high-efficiency off-target mutations in mice in vivo. Cell Death Dis 9:1099

    Article  PubMed  PubMed Central  Google Scholar 

  • Au V, Li-Leger E, Raymant G, Flibotte S, Chen G, Martin K, Fernando L, Doell C, Rosell FI, Wang S et al. (2019) CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans. G3 Bethesda 9:135–144

  • Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F (2014) Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 24:142–153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Borycz J, Borycz JA, Kubów A, Lloyd V, Meinertzhagen IA (2008) Drosophila ABC transporter mutants white, brown and scarlet have altered contents and distribution of biogenic amines in the brain. J Exp Biol 211:3454–3466

    Article  CAS  PubMed  Google Scholar 

  • Bruce HS, Patel NH (2022) The carapace and other novel structures evolved via the cryptic persistence of serial homologs. Curr Biol 32:3792–3799

    Article  CAS  PubMed  Google Scholar 

  • Carballar-Lejarazú R, Tushar T, Pham TB, James AA (2021) Microinjection method for Anopheles gambiae embryos. JoVE J Vis Exp 7:e62591

  • Chen X, Schulz-Trieglaff O, Shaw R, Barnes B, Schlesinger F, Kallberg M, Cox AJ, Kruglyak S, Saunders CT (2016) Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics 32:1220–1222

    Article  CAS  PubMed  Google Scholar 

  • Colbourne JK, Hebert PDN (1996) The systematics of North American Daphnia (Crustacea: Anomopoda): A molecular phylogenetic approach. Philos Trans R Soc Lond B Biol Sci 351:349–360

    Article  CAS  PubMed  Google Scholar 

  • Colbourne JK, Pfrender ME, Gilbert D, Thomas WK, Tucker A, Oakley TH, Tokishita S, Aerts A, Arnold GJ, Basu MK et al. (2011) The ecoresponsive genome of Daphnia pulex. Science 331:555–561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, Hsu PD, Wu XB, Jiang WY, Marraffini LA et al. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Crawford K, Diaz Quiroz JF, Koenig KM, Ahuja N, Albertin CB, Rosenthal JJC (2020) Highly Efficient Knockout of a Squid Pigmentation Gene. Curr Biol 30:3484–3490 e3484

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Curcio M, Bradke F (2018) Axon Regeneration in the Central Nervous System: Facing the Challenges from the Inside. Annu Rev Cell Dev Biol 34:495–521

    Article  CAS  PubMed  Google Scholar 

  • Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM et al. (2021) Twelve years of SAMtools and BCFtools. Gigascience 10:giab008

    Article  PubMed  PubMed Central  Google Scholar 

  • Davies B (2019) The technical risks of human gene editing. Hum Reprod 34:2104–2111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21

    Article  CAS  PubMed  Google Scholar 

  • Ebert D (2022) Daphnia as a versatile model system in ecology and evolution. Evodevo 13:16

    Article  PubMed  PubMed Central  Google Scholar 

  • Evans JM, Day JP, Cabrero P, Dow JAT, Davies SA (2008) A new role for a classical gene: white transports cyclic GMP. J Exp Biol 211:890–899

    Article  CAS  PubMed  Google Scholar 

  • Evans T (2006) Transformation and microinjection. In Transformation and microinjection, https://doi.org/10.1895/wormbook.1.108.1. The C. elegans Research Community.

  • Ewart GD, Cannell D, Cox GB, Howells AJ (1994) Mutational analysis of the traffic ATPase (ABC) transporters involved in uptake of eye pigment precursors in Drosophila melanogaster. Implications for structure-function relationships. J Biol Chem 269:10370–10377

    Article  CAS  PubMed  Google Scholar 

  • Flynn JM, Chain FJJ, Schoen DJ, Cristescu ME (2017) Spontaneous mutation accumulation in Daphnia pulex in selection-free vs. competitive environments. Mol Biol Evol 34:160–173

    Article  CAS  PubMed  Google Scholar 

  • Fu YF, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harris KDM, Bartlett NJ, Lloyd VK (2012) Daphnia as an Emerging Epigenetic Model Organism. Genet Res Int 2012:147892

    PubMed  PubMed Central  Google Scholar 

  • Hefferin ML, Tomkinson AE (2005) Mechanism of DNA double-strand break repair by non-homologous end joining. DNA Repair 4:639–648

    Article  CAS  PubMed  Google Scholar 

  • Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB et al. (2015) Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33:985–989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hiruta C, Kakui K, Tollefsen KE, Iguchi T (2018) Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex. Genes Cells 23:494–502

    Article  CAS  PubMed  Google Scholar 

  • Hiruta C, Nishida C, Tochinai S (2010) Abortive meiosis in the oogenesis of parthenogenetic Daphnia pulex. Chromosome Res 18:833–840

    Article  CAS  PubMed  Google Scholar 

  • Hiruta C, Toyota K, Miyakawa H, Ogino Y, Miyagawa S, Tatarazako N, Shaw JR, Iguchi T (2013) Development of a microinjection system for RNA interference in the water flea Daphnia pulex. BMC Biotechnol 13:96

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Höijer I, Emmanouilidou A, Östlund R, van Schendel R, Bozorgpana S, Tijsterman M, Feuk L, Gyllensten U, den Hoed M, Ameur A (2022) CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nat Commun 13:627

    Article  PubMed  PubMed Central  Google Scholar 

  • Huang S, Huang X (2023) A massively parallel approach for assessing CRISPR off-targets in vitro. Cell Rep. Methods 3:100561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huynh TV, Hall AS, Xu S (2023) The Transcriptomic Signature of Cyclical Parthenogenesis. Genome Biol Evol 15:evad122.

  • Ismail NIB, Kato Y, Matsuura T, Gómez-Canela C, Barata C, Watanabe H (2021) Reduction of histamine and enhanced spinning behavior of Daphnia magna caused by scarlet mutant. Genesis 59:e23403

  • Ismail NIB, Kato Y, Matsuura T, Watanabe H (2018) Generation of white-eyed Daphnia magna mutants lacking scarlet function. PLoS One 13:e0205609

    Article  PubMed  PubMed Central  Google Scholar 

  • Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337:816–821

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kato Y, Matsuura T, Watanabe H (2012) Genomic Integration and Germline Transmission of Plasmid Injected into Crustacean Daphnia magna Eggs. Plos One 7:e45318.

  • Kato Y, Perez CAG, Ishak NSM, Nong QD, Sudo Y, Matsuura T, Wada T, Watanabe H (2018) A 5 ‘ UTR-Overlapping LncRNA Activates the Male-Determining Gene doublesex1 in the Crustacean Daphnia magna. Curr Biol 28:1811–1817

    Article  CAS  PubMed  Google Scholar 

  • Kato Y, Shiga Y, Kobayashi K, Tokishita S, Yamagata H, Iguchi T, Watanabe H (2011) Development of an RNA interference method in the cladoceran crustacean Daphnia magna. Dev Genes Evol 220:337–345

    Article  CAS  PubMed  Google Scholar 

  • Keith N, Tucker AE, Jackson CE, Sung W, Lucas Lledo JI, Schrider DR, Schaack S, Dudycha JL, Ackerman M, Younge AJ et al. (2016) High mutational rates of large-scale duplication and deletion in Daphnia pulex. Genome Res 26:60–69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kilham SS, Kreeger DA, Lynn SG, Goulden CE, Herrera L (1998) COMBO: a defined freshwater culture medium for algae and zooplankton. Hydrobiologia 377:147–159

    Article  CAS  Google Scholar 

  • Kim S, Kim D, Cho SW, Kim J, Kim JS (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Koenig MK, Hodgeman R, Riviello JJ, Chung W, Bain J, Chiriboga CA, Ichikawa K, Osaka H, Tsuji M, Gibson KM et al. (2017) Phenotype of GABA-transaminase deficiency. Neurology 88:1919–1924

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumagai H, Nakanishi T, Matsuura T, Kato Y, Watanabe H (2017) CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna. Plos One 12:e0186112

    Article  PubMed  PubMed Central  Google Scholar 

  • Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26:589–595

    Article  PubMed  PubMed Central  Google Scholar 

  • Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed  PubMed Central  Google Scholar 

  • Liang F, Han M, Romanienko PJ, Jasin M (1998) Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci USA 95:5172–5177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liao Y, Smyth GK, Shi W (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923–930

    Article  CAS  PubMed  Google Scholar 

  • Louro P, Ramos L, Robalo C, Cancelinha C, Dinis A, Veiga R, Pina R, Rebelo O, Pop A, Diogo L et al. (2016) Phenotyping GABA transaminase deficiency: a case description and literature review. J Inherit Metab Dis 39:743–747

    Article  CAS  PubMed  Google Scholar 

  • Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550

    Article  PubMed  PubMed Central  Google Scholar 

  • Lynch M, Gutenkunst R, Ackerman M, Spitze K, Ye Z, Maruki T, Jia Z (2017) Population Genomics of Daphnia pulex. Genetics 206:315–332

    Article  CAS  PubMed  Google Scholar 

  • Mahato S, Morita S, Tucker AE, Liang X, Jackowska M, Friedrich M, Shiga Y, Zelhof AC (2014) Common transcriptional mechanisms for visual photoreceptor cell differentiation among Pancrustaceans. PLoS Genet 10:e1004484

    Article  PubMed  PubMed Central  Google Scholar 

  • Maresca M, Lin VG, Guo N, Yang Y (2013) Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res 23:539–546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miner BE, De Meester L, Pfrender ME, Lampert W, Hairston NG (2012) Linking genes to communities and ecosystems: Daphnia as an ecogenomic model. Proc R Soc Biol Sci B 279:1873–1882

    Article  Google Scholar 

  • Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, Khokha MK, Doudna JA, Giraldez AJ (2017) CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun 8:2024

    Article  PubMed  PubMed Central  Google Scholar 

  • Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35:W182–W185

    Article  PubMed  PubMed Central  Google Scholar 

  • Nakanishi T, Kato Y, Matsuura T, Watanabe H (2016) TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna. Combining Mult Hypothesis Sci Rep. 6:36252

    CAS  Google Scholar 

  • Ojima Y (1958) A cytological study on the development and maturation of the parthenogenetic and sexual eggs of Daphnia pulex. Kwansei Gakuin Univ Ann Stud 6:123–171

    Google Scholar 

  • Owens DDG, Caulder A, Frontera V, Harman JR, Allan AJ, Bucakci A, Greder L, Codner GF, Hublitz P, McHugh PJ et al. (2019) Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic Acids Res 47:7402–7417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Parkinson MJ, Lilley DM (1997) The junction-resolving enzyme T7 endonuclease I: quaternary structure and interaction with DNA. J Mol Biol 270:169–178

    Article  CAS  PubMed  Google Scholar 

  • Peippo M, Ignatius J (2011) Pitt-Hopkins Syndrome. Pitt-Hopkins Syndr 2:171–180

    Google Scholar 

  • Rasys AM, Park S, Ball RE, Alcala AJ, Lauderdale JD, Menke DB (2019) CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes. Cell Rep. 28:2288–2292.e2283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Riesenberg S, Kanis P, Macak D, Wollny D, Dusterhoft D, Kowalewski J, Helmbrecht N, Maricic T, Paabo S (2023) Efficient high-precision homology-directed repair-dependent genome editing by HDRobust. Nat Methods 20:1388–1399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ringrose L (2009) Transgenesis in Drosophila melanogaster. Methods Mol Biol 561:3–19

    Article  CAS  PubMed  Google Scholar 

  • Rivera AS, Pankey MS, Plachetzki DC, Villacorta C, Syme AE, Serb JM, Omilian AR, Oakley TH (2010) Gene duplication and the origins of morphological complexity in pancrustacean eyes, a genomic approach. BMC Evol Biol 10:123

    Article  PubMed  PubMed Central  Google Scholar 

  • Rodgers K, McVey M (2016) Error-Prone Repair of DNA Double-Strand Breaks. J Cell Physiol 231:15–24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schimmel J, Munoz-Subirana N, Kool H, van Schendel R, van der Vlies S, Kamp JA, de Vrij FMS, Kushner SA, Smith GCM, Boulton SJ et al. (2023) Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways. Cell Rep. 42:112019

    Article  CAS  PubMed  Google Scholar 

  • Sekelsky J (2017) DNA Repair in Drosophila: Mutagens, Models, and Missing Genes. Genetics 205:471–490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sfeir A, Symington LS (2015) Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway? Trends Biochem Sci 40:701–714

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sharma A, Pham MN, Reyes JB, Chana R, Yim WC, Heu CC, Kim D, Chaverra-Rodriguez D, Rasgon JL, Harrell RA et al. (2022) Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control. iScience, https://doi.org/10.1016/j.isci.2022.103781

  • Sharon E, Chen SA, Khosla NM, Smith JD, Pritchard JK, Fraser HB (2018) Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing. Cell 175:544–557.e516

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shaw JR, Colbourne JK, Davey JC, Glaholt SP, Hampton TH, Chen CY, Folt CL, Hamilton JW (2007) Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins. BMC Genomics 8:477

    Article  PubMed  PubMed Central  Google Scholar 

  • Shin HY, Wang C, Lee HK, Yoo KH, Zeng X, Kuhns T, Yang CM, Mohr T, Liu C, Hennighausen L (2017) CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. Nat Commun 8:15464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shirai Y, Piulachs M-D, Belles X, Daimon T (2022) DIPA-CRISPR is a simple and accessible method for insect gene editing. Cell Rep Methods 2:100215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, Chen YY, Blaeschke F, Shifrut E et al. (2023) High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat Biotechnol 41:521–531

    Article  CAS  PubMed  Google Scholar 

  • Snyman M, Huynh TV, Smith MT, Xu S (2021) The genome-wide rate and spectrum of EMS-induced heritable mutations in the microcrustacean Daphnia: on the prospect of forward genetics. Heredity 127:535–545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Snyman M, Xu S (2023) Transcriptomics and the origin of obligate parthenogenesis. Heredity 131:119–129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Toyota K, Miyagawa S, Ogino Y, Iguchi T (2016) Microinjection-based RNA interference method in the water flea, Daphnia pulex and Daphnia magna. In RNA Interference. IntechOpen.

  • Trible W, Olivos-Cisneros L, McKenzie SK, Saragosti J, Chang NC, Matthews BJ, Oxley PR, Kronauer DJC (2017) orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants. Cell 170:727–735.e710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang H, Xu S (2021) Something old, something new, a DNA preparation procedure for long-read genomic sequencing. Genome 65:53–56

    Article  Google Scholar 

  • Wersebe MJ, Weider LJ (2023) Resurrection genomics provides molecular and phenotypic evidence of rapid adaptation to salinization in a keystone aquatic species. Proc Natl Acad Sci USA 120:e2217276120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu S, Huynh TV, Snyman M (2022) The transcriptomic signature of obligate parthenogenesis. Heredity 128:132–138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu S, Omilian AR, Cristescu ME (2011) High rate of large-scale hemizygous deletions in asexually propagating Daphnia: omplications for the evolution of sex. Mol Biol Evol 28:335–342

    Article  CAS  PubMed  Google Scholar 

  • Xu S, Pham TP, Neupane S (2020) Delivery methods for CRISPR/Cas9 gene editing in crustaceans. Mar Life Sci Technol 2:1–5

    Article  CAS  PubMed  Google Scholar 

  • Xu S, Spitze K, Ackerman MS, Ye Z, Bright L, Keith N, Jackson CE, Shaw JR, Lynch M (2015) Hybridization and the origin of contagious asexuality in Daphnia pulex. Mol Biol Evol 32:3215–3225

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ye Z, Pfrender ME, Lynch M (2023) Evolutionary Genomics of Sister Species Differing in Effective Population Sizes and Recombination Rates. Genome Biol Evol 15:evad202

  • Ye Z, Xu S, Spitze K, Asselman J, Jiang X, Ackerman MS, Lopez J, Harker B, Raborn RT, Thomas WK et al. (2017) A new reference genome assembly for the microcrustacean Daphnia pulex. G3 Bethesda 7:1405–1416

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zaffagnini F, Sabelli B (1972) Karyologic observations on the maturation of the summer and winter eggs of Daphnia pulex and Daphnia middendorffiana. Chromosoma 36:193–203

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank J. Dittmer and P. Wright for their assistance with swimming behavior experiments. We also thank Michael Pfrender for his comments on an early draft of this manuscript. This work is supported by NIH grant R35GM133730, NSF CAREER grant MCB-2042490/2348390, NSF EDGE grant 2220695/2324639 to SX.

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Author notes

  1. Hongjun Wang

    Present address: Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX, 75235, USA

  2. Marelize Snyman

    Present address: Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, TX, 75235, USA

  3. These authors contributed equally: Swatantra Neupane, Hongjun Wang, Thinh Phu Pham.

Authors and Affiliations

  1. Division of Biological Sciences, University of Missouri, Columbia, MO, 65211, USA

    Sen Xu, Swatantra Neupane, Trung V. Huynh & Li Wang

  2. Department of Biology, University of Texas at Arlington, Arlington, TX, 76019, USA

    Hongjun Wang, Thinh Phu Pham & Marelize Snyman

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SX designed research, performed experiments, analyzed data, and wrote the manuscript. SN, HW, and TPP performed microinjection experiments, analyzed mutant and genomic data, and contributed to the writing of the manuscript. MS performed RNA-seq experiments, analyzed RNA-seq data, and contributed to the writing of manuscript. TH analyzed knock-in mutant genomic sequences. LW performed behavior experiments of scarlet mutants and analyzed data.

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Correspondence to Sen Xu.

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Xu, S., Neupane, S., Wang, H. et al. The mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system. Heredity 134, 221–233 (2025). https://doi.org/10.1038/s41437-025-00750-4

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