Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system

  • Xiuwen Xu
  • Xiaojuan CaoEmail author
  • Jian Gao
Original Paper


CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species.


Paramisgurnus dabryanus Tyrosinase gene Cloning and expression Gene knock-out CRISPR/Cas9 system Skin pigmentation loss 


Author contributions

XX, XC and JG performed the experiments. XC designed the experiment. JG performed the bioinformatics analysis. XX and XC prepared the manuscript. All authors read and approved the final version of the manuscript.


This study was financially supported by National Key R&D Program of China (Project Number: 2018YFD0900200) and the Fundamental Research Funds for the Central Universities of China (Project Number: 2662015PY033).

Compliance with ethical standards

Conflict of interest

The authors have declared that no conflicts of interests exist.

Ethical approval

This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Huazhong Agricultural University. All efforts were made to minimize suffering.


  1. Berthelot C, Brunet F, Chalopin D, Juanchich A, Bernard M, Noël B, Bento P, Da SC, Labadie K, Alberti A (2014) The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates. Nat Commun 5:3657CrossRefGoogle Scholar
  2. Boonanuntanasarn S, Yoshizaki G, Iwai K, Takeuchi T (2004) Molecular cloning, gene expression in albino mutants and gene knockdown studies of tyrosinase mRNA in rainbow trout. Pigment Cell Res 17:413–421CrossRefGoogle Scholar
  3. Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52CrossRefGoogle Scholar
  4. Camacho-Hübner A, Rossier A, Beermann F (2000) The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse. Genesis 28:99–105CrossRefGoogle Scholar
  5. Camacho-Hübner A, Richard C, Beermann F (2002) Genomic structure and evolutionary conservation of the tyrosinase gene family from Fugu. Gene 285:59–68CrossRefGoogle Scholar
  6. Chen G, Huang S, Gao J, Bai X, Wang W, Cao X (2015) Development and characterization of microsatellite markers via cross-species amplification of Paramisgurnus dabryanus. Genet Mol Res 14:5694–5698CrossRefGoogle Scholar
  7. Chen H, Wang J, Du J, Si Z, Yang H, Xu X, Wang C (2019) ASIP disruption via CRISPR/Cas9 system induces black patches dispersion in Oujiang color common carp. Aquaculture 498:230–235CrossRefGoogle Scholar
  8. Cho YS, Kim BS, Dong SK, Nam YK (2012) Modulation of warm-temperature-acclimation-associated 65-kDa protein genes (Wap65-1 and Wap65-2) in mud loach (Misgurnus mizolepis, Cypriniformes) liver in response to different stimulatory treatments. Fish Shellfish Immunol 32:662–669CrossRefGoogle Scholar
  9. Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232CrossRefGoogle Scholar
  10. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hus PD, Wu X, Jing W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefGoogle Scholar
  11. Daer RM, Cutts JP, Brafman DA, Haynes KA (2017) The impact of chromatin dynamics on Cas9-mediated genome editing in human cells. ACS Synth Biol 6:428–438CrossRefGoogle Scholar
  12. Davies FCJ, Hope JE, Mclachlan F, Nunez F, Doig J, Bengani H, Smith C, Abbott CM (2017) Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice. Scientific reports 7:46019CrossRefGoogle Scholar
  13. Dong Z, Ge J, Li K, Xu Z, Liang D, Li J, Li J, Jia W, Li Y, Dong X (2011) Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco) using engineered zinc finger nucleases. PLoS ONE 6:e28897CrossRefGoogle Scholar
  14. Dooley CM, Schwarz H, Mueller KP, Mongera A, Konantz M, Neuhauss SCF, Nüsslein-Volhard C, Geisler R (2013) Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease. Pigment Cell Melanoma Res 26:205–217CrossRefGoogle Scholar
  15. Edvardsen RB, Leininger S, Kleppe L, Kai OS, Wargelius A (2014) Targeted mutagenesis in atlantic salmon (Salmo salar L.) using the CRISPR/Cas9 system induces complete knockout individuals in the F0 generation. PloS ONE 9:e108622Google Scholar
  16. Elaswad A, Khalil K, Ye Z, Liu Z, Liu S, Peatman E, Odin R, Vo K, Drescher D, Gosh K (2018) Effects of CRISPR/Cas9 dosage on TICAM1 and RBL gene mutation rate, embryonic development, hatchability and fry survival in channel catfish. Sci Rep 8:16499CrossRefGoogle Scholar
  17. Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, Luo K (2015) Efficient CRISPR/Cas9-mediated targeted mutagenesis in populus in the first generation. Sci Rep 5:12217CrossRefGoogle Scholar
  18. Ghosh SK, Ghosh B, Chakrabarti P (2011) Fine anatomical structures of the intestine in relation to respiratory function of an air-breathing loach, Lepidocephalichthys guntea (Actinopterygii: Cypriniformes: Cobitidae). Acta Ichthyol Piscat 41:1–5CrossRefGoogle Scholar
  19. Goncalves A, Castro L, Pereira-Wilson C, Coimbra J, Wilson J (2007) Is there a compromise between nutrient uptake and gas exchange in the gut of Misgurnus anguillicaudatus, an intestinal air-breathing fish? Comp Biochem Physiol D-Genomics Proteom 2:345–355CrossRefGoogle Scholar
  20. Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O’Connorgiles KM (2013) Genome engineering of drosophila with the CRISPR RNA-guided Cas9. Nucl Genet 194:1029–1035CrossRefGoogle Scholar
  21. Greenhill ER, Rocco A, Vibert L, Nikaido M, Kelsh RN (2011) An iterative genetic and dynamical modelling approach identifies novel features of the gene regulatory network underlying melanocyte development. PLoS Genet 7:e1002265CrossRefGoogle Scholar
  22. Huang S, Cao X, Tian X (2016a) Transcriptomic analysis of compromise between air-breathing and nutrient uptake of posterior intestine in loach (Misgurnus anguillicaudatus), an air-breathing fish. Mar Biotechnol 18:521–533CrossRefGoogle Scholar
  23. Huang S, Cao X, Tian X, Wang W (2016b) High-throughput sequencing identifies microRNAs from posterior intestine of loach (Misgurnus anguillicaudatus) and their response to intestinal air-breathing inhibition. PLoS ONE 11:e0149123CrossRefGoogle Scholar
  24. Iida A, Inagaki H, Suzuki M, Wakamatsu Y, Hori H, Koga A (2004) The tyrosinase gene of the i(b) albino mutant of the medaka fish carries a transposable element insertion in the promoter region. Pigment Cell Res 17:158–164CrossRefGoogle Scholar
  25. Jao LE, Wente SR, Chen W (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 110:13904–13909CrossRefGoogle Scholar
  26. Jensen ED, Ferreira R, Jakočiūnas T, Arsovska D, Zhang J, Ding L, Smith JD, David F, Nielsen J, Jensen MK (2017) Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies. Microb Cell Fact 16:46CrossRefGoogle Scholar
  27. Jiang D, Chen J, Fan Z, Tan D, Zhao J, Shi H, Liu Z, Tao W, Li M, Wang D (2017) CRISPR/Cas9-induced disruption of wt1a and wt1b reveals their different roles in kidney and gonad development in Nile tilapia. Dev Biol 428:63–73CrossRefGoogle Scholar
  28. Katic I, Großhans H (2013) Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans. Genetics 195:1173–1176CrossRefGoogle Scholar
  29. Kishimoto K, Washio Y, Yoshiura Y, Toyoda A, Ueno T, Fukuyama H, Kato K, Kinoshita M (2018) Production of a breed of red sea bream Pagrus major with an increase of skeletal muscle muss and reduced body length by genome editing with CRISPR/Cas9. Aquaculture 495:415–427CrossRefGoogle Scholar
  30. Koga A, Wakamatsu Y, Jin K, Hori H (1999) Oculocutaneous albinism in the i6 mutant of the medaka fish is associated with a deletion in the tyrosinase gene. Pigment Cell Res 12:252–258CrossRefGoogle Scholar
  31. Kurokura H, Hirano R, Tomita M, Iwahashi M (1984) Cryopreservation of carp sperm. Aquaculture 37:267–273CrossRefGoogle Scholar
  32. Li YJ, Zhang MZ, Qian C, Gao M, Arai K (2012) Fertility and ploidy of gametes of diploid, triploid and tetraploid loaches, Misgurnus anguillicaudatus, in China. J Appl Ichthyol 28:900–905CrossRefGoogle Scholar
  33. Li M, Yang H, Zhao J, Fang L, Shi H, Li M, Sun Y, Zhang X, Jiang D, Zhou L (2014) Efficient and heritable gene targeting in tilapia by CRISPR/Cas9. Genetics 197:591–599CrossRefGoogle Scholar
  34. Li M, Feng R, Ma H, Dong R, Liu Z, Jiang W, Tao W, Wang D (2016) Retinoic acid triggers meiosis initiation via stra8 -dependent pathway in Southern catfish, Silurus meridionalis. Gen Comp Endocrinol 232:191–198CrossRefGoogle Scholar
  35. Lin CY, Su YH (2016) Genome editing in sea urchin embryos by using a CRISPR/Cas9 system. Dev Biol 409:420–428CrossRefGoogle Scholar
  36. Luo YJ, Su YH (2012) Opposing nodal and BMP signals regulate left–right asymmetry in the sea urchin larva. PLoS Biol 10:e1001402CrossRefGoogle Scholar
  37. Luo W, Cao X, Xu X, Huang S, Liu C, Tomljanovic T (2016a) Developmental transcriptome analysis and identification of genes involved in formation of intestinal air-breathing function of Dojo loach, Misgurnus anguillicaudatus. Sci Rep 6:31845CrossRefGoogle Scholar
  38. Luo W, Liang X, Huang S, Cao X (2016b) Molecular cloning, expression analysis and miRNA prediction of vascular endothelial growth factor A (VEGFAa and VEGFAb) in pond loach Misgurnus anguillicaudatus, an air-breathing fish. Comp Biochem Physiol B: Biochem Mol Biol 202:39–47CrossRefGoogle Scholar
  39. Ma J, Fan Y, Zhou Y, Liu W, Jiang N, Zhang J, Zeng L (2018) Efficient resistance to grass carp reovirus infection in JAM-A knockout cells using CRISPR/Cas9. Fish Shellfish Immunol 76:206–215CrossRefGoogle Scholar
  40. Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefGoogle Scholar
  41. Mcmahon BR, Burggren WW (1987) Respiratory physiology of intestinal air breathing in the teleost fish Misgurnus Anguillicaudatus. J Exp Biol 133:371–393Google Scholar
  42. Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12:982–988CrossRefGoogle Scholar
  43. Müller G, Ruppert S, Schmid E, Schütz G (1988) Functional analysis of alternatively spliced tyrosinase gene transcripts. EMBO J 7:2723–2730CrossRefGoogle Scholar
  44. Nakayama T, Fish MB, Fisher M, Oomenhajagos J, Thomsen GH, Grainger RM (2013) Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis 51:835–843CrossRefGoogle Scholar
  45. Nishioka K (1978) Particulate tyrosinase of human malignant melanoma. Eur J Biochem 85:137–146CrossRefGoogle Scholar
  46. Nishitani C, Hirai N, Komori S, Wada M, Okada K, Osakabe K, Yamamoto T, Osakabe Y (2016) Efficient genome editing in apple using a CRISPR/Cas9 system. Sci Rep 6:31481CrossRefGoogle Scholar
  47. Page-McCaw PS, Chung SA, Roeser T, Staub W, Finger Baier KC, Korenbrot JI, Baier H (2004) Retinal network adaptation to bright light requires tyrosinase. Nat Neurosci 7:1329–1336CrossRefGoogle Scholar
  48. Sato S, Masuya H, Numakunai T, Satoh N, Ikeo K, Gojobori T, Tamura K, Ide H, Takeuchi T, Yamamoto H (1997) Ascidian tyrosinase gene: its unique structure and expression in the developing brain. Dev Dyn 208:363–374CrossRefGoogle Scholar
  49. Sato S, Tanaka M, Miura H, Ikeo K, Gojobori T, Takeuchi T, Yamamoto H (2001) Functional conservation of the promoter regions of vertebrate tyrosinase genes. J Investig Dermatol Symp Proc 6:10–18CrossRefGoogle Scholar
  50. Smith JJ, Kuraku S, Holt C, Saukaspengler T, Ning J, Campbell MS, Yandell MD, Manousaki T, Meyer A, Bloom OE (2013) Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution. Nat Genet 45:415–421CrossRefGoogle Scholar
  51. Uusi-Mäkelä MIE, Barker HR, Bäuerlein CA, Häkkinen T, Nykter M, Rämet M (2018) Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio). PLoS ONE 13:e0196238CrossRefGoogle Scholar
  52. Wang W, Hu H, Sun X, Niu C (2012) Analysis of tyrosinase gene and tissue expression in five different strains of Koi carp (Cyprinus carpio Koi). J Fish China 36(11):1658–1666CrossRefGoogle Scholar
  53. Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338CrossRefGoogle Scholar
  54. Won M, Dawid IB (2017) PCR artifact in testing for homologous recombination in genomic editing in zebrafish. PLoS ONE 12:e0172802CrossRefGoogle Scholar
  55. Xiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30:1180–1182CrossRefGoogle Scholar
  56. Xin HH (2015) Application of CRISPR/Cas9 system in silkworm gene function studies. Doctor’s thesis, Zhejiang University, Hangzhou, ChinaGoogle Scholar
  57. Yano A, Nicol B, Jouanno E, Quillet E, Fostier A, Guyomard R, Guiguen Y (2013) The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids. Evol Appl 6:486–496CrossRefGoogle Scholar
  58. Yarrington RM, Verma S, Schwartz S, Trautman JK, Carroll D (2018) Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo. Proc Natl Acad Sci 115:9351–9358CrossRefGoogle Scholar
  59. Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G (2013) Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195:289–291CrossRefGoogle Scholar
  60. Zamanian M, Andersen EC (2016) Prospects and challenges of CRISPR/Cas genome editing for the study and control of neglected vector-borne nematode diseases. FEBS J 283:3204–3221CrossRefGoogle Scholar
  61. Zu Y, Zhang X, Ren J, Dong X, Zhu Z, Jia L, Zhang Q, Li W (2016) Biallelic editing of a lamprey genome using the CRISPR/Cas9 system. Scientific reports 6:23496CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of AgricultureHuazhong Agricultural UniversityWuhanPeople’s Republic of China
  2. 2.Hubei Provincial Engineering Laboratory for Pond AquacultureWuhanPeople’s Republic of China

Personalised recommendations