Abstract
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.
References
Ansai S, Kinoshita M (2014) Targeted mutagenesis using CRISPR/Cas system in medaka. Biology Open 3(5):362–371. https://doi.org/10.1242/bio.20148177
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. https://doi.org/10.1534/genetics.114.169730
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. https://doi.org/10.1038/nature09164
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. https://doi.org/10.1002/anie.201206797
Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77(1):71–94. http://www.genetics.org/content/genetics/77/1/71.full.pdf
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. https://doi.org/10.1016/j.cell.2012.12.013
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. https://doi.org/10.1093/nar/gkt805
Chiu H, Schwartz HT, Antoshechkin I, Sternberg PW (2013) Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics 195(3):1167–1171. https://doi.org/10.1534/genetics.113.155879
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. https://doi.org/10.1534/genetics.113.155853
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. https://doi.org/10.1126/science.1231143
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. https://doi.org/10.1038/nmeth.2641
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. https://doi.org/10.1038/ng.227
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. https://doi.org/10.1534/genetics.118.301532
Eizinger A, Sommer RJ (1997) The homeotic gene lin-39 and the evolution of nematode epidermal cell fates. Science 278(5337):452–455. https://doi.org/10.1126/science.278.5337.452
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. https://doi.org/10.1016/j.chembiol.2018.04.004
Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31(7):397-405 https://doi.org/10.1016/j.tibtech.2013.04.004
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. https://doi.org/10.1073/pnas.0708406105
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278. https://doi.org/10.1016/j.cell.2014.05.010
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. https://doi.org/10.1126/science.1225829
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. eLife 2:e00471. https://doi.org/10.7554/eLife.00471
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. https://doi.org/10.1002/gene.20084
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. https://doi.org/10.1534/genetics.114.166389
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
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. https://doi.org/10.1091/mbc.4.8.803
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. https://doi.org/10.1126/science.aav9856
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. https://doi.org/10.1534/genetics.113.155382
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. https://doi.org/10.1126/science.1232033
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. https://doi.org/10.1371/journal.pgen.1005146
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. https://doi.org/10.1098/rspb.2019.1089
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. https://doi.org/10.1007/s00427-018-0605-z
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). https://doi.org/10.1242/dev.166272
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. https://doi.org/10.1534/g3.117.300263
Park EC, Horvitz HR (1986) Mutations with dominant effects on the behavior and morphology of the nematode Caenorhabditis elegans. Genetics 113(4):821–852
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. https://doi.org/10.1016/j.cell.2013.09.054
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. https://doi.org/10.1186/s12864-016-2881-1
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. https://doi.org/10.1002/dvg.20499
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. https://doi.org/10.1038/ncomms12337
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. https://doi.org/10.1093/molbev/msx106
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
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. https://doi.org/10.1016/S0960-9822(02)00421-9
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.
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. https://doi.org/10.1534/genetics.113.156075
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. https://doi.org/10.1002/humu.21019
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482(7385):331–338. https://doi.org/10.1038/nature10886
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. https://doi.org/10.1242/jeb.118620
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. https://doi.org/10.1007/s00427-014-0486-8
Wittwer CT (2009) High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30(6):857–859. https://doi.org/10.1002/humu.20951
Acknowledgments
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 (www.editage.jp) for English language editing.
Funding
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.
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Nakayama, Ki., 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 230, 257–264 (2020). https://doi.org/10.1007/s00427-020-00651-y
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DOI: https://doi.org/10.1007/s00427-020-00651-y