Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus


CRISPR/Cas9 genome-editing methods are used to reveal functions of genes and molecular mechanisms underlying biological processes in many species, including nematodes. In evolutionary biology, the nematode Pristionchus pacificus is a satellite model and has been used to understand interesting phenomena such as phenotypic plasticity and self-recognition. In P. pacificus, CRISPR/Cas9-mediated mutations are induced by microinjecting a guide RNA (gRNA) and Cas9 protein into the gonads. However, mutant screening is laborious and time-consuming due to the absence of visual markers. In this study, we established a Co-CRISPR strategy by using a dominant roller marker in P. pacificus. We found that heterozygous mutations in Ppa-prl-1 induced the roller phenotype, which can be used as an injection marker. After the co-injection of Ppa-prl-1 gRNA, target gRNA, and the Cas9 protein, roller progeny and their siblings were examined using the heteroduplex mobility assay and DNA sequencing. We found that some of the roller and non-roller siblings had mutations at the target site. We used varying Cas9 concentrations and found that a higher concentration of Cas9 did not increase genome-editing events. The Co-CRISPR strategy promotes the screening for genome-editing events and will facilitate the development of new genome-editing methods in P. pacificus.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3


  1. Ansai S, Kinoshita M (2014) Targeted mutagenesis using CRISPR/Cas system in medaka. Biology Open 3(5):362–371.

  2. Arribere JA, Bell RT, Fu BXH, Artiles KL, Hartman PS, Fire AZ (2014) Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics 198:837–846.

  3. Bento G, Ogawa A, Sommer RJ (2010) Co-option of the hormone-signalling module dafachronic acid-DAF-12 in nematode evolution. Nature 466(7305):494–497.

  4. Bose N, Ogawa A, von Reuss SH, Yim JJ, Ragsdale EJ, Sommer RJ, Schroeder FC (2012) Complex small-molecule architectures regulate phenotypic plasticity in a nematode. Angew Chem Int Ed 51(50):12438–12443.

  5. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77(1):71–94.

  6. Bumbarger DJ, Riebesell M, Rödelsperger C, Sommer RJ (2013) System-wide rewiring underlies behavioral differences in predatory and bacterial-feeding nematodes. Cell 152(1–2):109–119.

  7. Chen C, Fenk LA, De Bono M (2013) Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination. Nucleic Acids Res 41(20):e193.

  8. Chiu H, Schwartz HT, Antoshechkin I, Sternberg PW (2013) Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics 195(3):1167–1171.

  9. Cho SW, Lee J, Carroll D, Kim JS, Lee J (2013) Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195(3):1177–1180.

  10. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823.

  11. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B (2013) Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nature Methods 10(10):1028-1034.

  12. Dieterich C, Clifton SW, Schuster LN, Chinwalla A, Delehaunty K, Dinkelacker I, Fulton L, Fulton R, Godfrey J, Minx P, Mitreva M, Roeseler W, Tian H, Witte H, Yang SP, Wilson RK, Sommer RJ (2008) The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism. Nat Genet 40(10):1193–1198.

  13. Dokshin GA, Ghanta KS, Piscopo KM, Mello CC (2018) Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans. GENETICS 210(3):781–787.

  14. Eizinger A, Sommer RJ (1997) The homeotic gene lin-39 and the evolution of nematode epidermal cell fates. Science 278(5337):452–455.

  15. Falcke JM, Bose N, Artyukhin AB, Rödelsperger C, Markov GV, Yim JJ, Grimm D, Claassen MH, Panda O, Baccile JA, Zhang YK, le HH, Jolic D, Schroeder FC, Sommer RJ (2018) Linking genomic and metabolomic natural variation uncovers nematode pheromone biosynthesis. Cell Chemical Biology 25(6):787–796.

  16. Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31(7):397-405

  17. Hong RL, Witte H, Sommer RJ (2008) Natural variation in Pristionchus pacificus insect pheromone attraction involves the protein kinase EGL-4. Proc Natl Acad Sci U S A 105(22):7779–7784.

  18. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278.

  19. 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(6096):816–821.

  20. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. eLife 2:e00471.

  21. Kenning C, Kipping I, Sommer RJ (2004) Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus. Genesis 40(3):176–183.

  22. Kim H, Ishidate T, Ghanta KS, Seth M, Conte D, Shirayama M, Mello CC (2014) A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics 197(4):1069–1080.

  23. Kramer JM, Johnson JJ (1993) Analysis of mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans. Genetics 135(4):1035–1045

  24. Levy AD, Yang J, Kramer JM (1993) Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology. Mol Biol Cell 4(8):803–817.

  25. Lightfoot, J. W., Martin Wilecki, C. R., Moreno, E., Susoy, V., Witte, H., & Sommer, R. J. (2019). Small peptide–mediated self-recognition prevents cannibalism in predatory nematodes. Science 364(6435), 86–89.

  26. Lo TW, Pickle CS, Lin S, Ralston EJ, Gurling M, Schartner CM, Bian Q, Doudna JA, Meyer BJ (2013) Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics 195(2):331–348.

  27. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826.

  28. Mayer MG, Rödelsperger C, Witte H, Riebesell M, Sommer RJ (2015) The orphan gene dauerless regulates dauer development and intraspecific competition in nematodes by copy number variation. PLoS Genet 11(6):1005146.

  29. Moreno E, Lightfoot JW, Lenuzzi M, Sommer RJ (2019) Cilia drive developmental plasticity and are essential for efficient prey detection in predatory nematodes. Proc R Soc B Biol Sci 286(1912):20191089.

  30. Namai S, Sugimoto A (2018) Transgenesis by microparticle bombardment for live imaging of fluorescent proteins in Pristionchus pacificus germline and early embryos. Dev Genes Evol 228(1):75–82.

  31. Namdeo S, Moreno E, Rödelsperger C, Baskaran P, Witte H, Sommer RJ (2018) Two independent sulfation processes regulate mouth-form plasticity in the nematode Pristionchus pacificus. Development 145(13).

  32. Okumura M, Wilecki M, & Sommer RJ (2017) Serotonin drives predatory feeding behavior via synchronous feeding rhythms in the nematode Pristionchus pacificus. G3, 7:3745–3755.

  33. Park EC, Horvitz HR (1986) Mutations with dominant effects on the behavior and morphology of the nematode Caenorhabditis elegans. Genetics 113(4):821–852

  34. Ragsdale EJ, Müller MR, Rödelsperger C, Sommer RJ (2013) A developmental switch coupled to the evolution of plasticity acts through a sulfatase. Cell 155(4):922–933.

  35. Samarut É, Lissouba A, Drapeau P (2016) A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using high resolution melting analysis. BMC Genomics 17:547.

  36. Schlager B, Wang X, Braach G, Sommer RJ (2009) Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus. Genesis 47(5):300–304.

  37. Serobyan V, Xiao H, Namdeo S, Rödelsperger C, Sieriebriennikov B, Witte H, Röseler W, Sommer RJ (2016) Chromatin remodelling and antisense-mediated up-regulation of the developmental switch gene eud-1 control predatory feeding plasticity. Nat Commun 7:12337.

  38. Sieriebriennikov, B., Markov, G. V, Witte, H., & Sommer, R. J. (2017). The role of DAF-21/Hsp90 in mouth-form plasticity in Pristionchus pacificus. Mol Biol Evol, 34(7):1644–1653.

  39. Sommer RJ, Carta L, Kim SY, Sternberg PW (1996) Morphological, genetic and molecular description of Pristionchus pacificus sp. n. (Nematoda:Neodiplogastridae). Fundamental and Applied Nematology 19(6):511–521

  40. Sommer RJ, Sternberg PW (1996) Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis. Curr Biol 6(1):52–59.

  41. Sommer RJ, Sternberg PW, Srinivasan J, Rödelsperger C, Schroeder FC, et al. (2015) Pristionchus pacificus: a nematode model for comparative and evolutionary biology edited by Sommer, R. J.. Brill, Leiden, The Netherlands.

  42. Tzur YB, Friedland AE, Nadarajan S, Church GM, Calarco JA, Colaiácovo MP (2013) Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system. Genetics 195(3):1181–1185.

  43. Vossen RHAM, Aten E, Roos A, Den Dunnen JT (2009) High-resolution melting analysis (HRMA)-more than just sequence variant screening. Hum Mutat 30(6):860–866.

  44. Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482(7385):331–338.

  45. Wilecki M, Lightfoot JW, Susoy V, Sommer RJ (2015) Predatory feeding behaviour in Pristionchus nematodes is dependent on phenotypic plasticity and induced by serotonin. J Exp Biol 218:1306–1313.

  46. Witte H, Moreno E, Rödelsperger C, Kim J, Kim J-S, Streit A, Sommer RJ (2015) Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus. Dev Genes Evol 225:55–62.

  47. Wittwer CT (2009) High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30(6):857–859.

Download references


We would like to thank Dr. Keisuke Nakajima (Institute for Amphibian Biology, Hiroshima University) and Mr. Nobuo Yamaguchi (Natural Science Center for Basic Research and Development, Hiroshima University) for helping with microchip electrophoresis. We also thank all the members of Chihara laboratory for their kind support and Editage ( for English language editing.


This work was supported by JSPS KAKENHI Grant Number 18K14716 to MO and 18H05369 to TC. This work was also supported by Tomizawa Jun-ichi & Keiko Fund of Molecular Biology Society of Japan for Young Scientist and AMED under Grant Number JP19gm6310003 to MO, and Toray Science Foundation, The Frontier Development Program for Genome Editing, and Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers JPMXS05S2900002 to TC.

Author information

Correspondence to Misako Okumura.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by Ralf Sommer

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nakayama, K., Ishita, Y., Chihara, T. et al. Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus. Dev Genes Evol (2020).

Download citation


  • Pristionchus pacificus
  • CRISPR/Cas9
  • Co-injection marker
  • Microchip electrophoresis