CRISPR Knockouts in Ciona Embryos

  • Shashank Gandhi
  • Florian Razy-Krajka
  • Lionel ChristiaenEmail author
  • Alberto StolfiEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1029)


Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a revolutionary tool for fast and efficient targeted gene knockouts and genome editing in almost any organism. The laboratory model tunicate Ciona is no exception. Here, we describe our latest protocol for the design, implementation, and evaluation of successful CRISPR/Cas9-mediated gene knockouts in somatic cells of electroporated Ciona embryos. Using commercially available reagents, publicly accessible plasmids, and free web-based software applications, any Ciona researcher can easily knock out any gene of interest in their favorite embryonic cell lineage.


Genome editing Targeted mutagenesis Somatic gene knockout sgRNAs Tunicates Chordates 



Research in the laboratory of L.C. is supported by R01 awards HL108643 and GM096032 from the NIH/NHLBI and NIH/NIGMS respectively; and by grant 15CVD01 from the Leducq Foundation. A.S. is supported by R00 award HD084814 from the NIH/NICHD.


  1. Abdul-Wajid S, Morales-Diaz H, Khairallah SM, Smith WC (2015) T-type Calcium Channel regulation of neural tube closure and EphrinA/EPHA expression. Cell Rep 13:829–839CrossRefPubMedPubMedCentralGoogle Scholar
  2. Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–573CrossRefPubMedPubMedCentralGoogle Scholar
  3. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712CrossRefPubMedGoogle Scholar
  4. Beerli RR, Barbas CF (2002) Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 20:135–141CrossRefPubMedGoogle Scholar
  5. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764–764CrossRefPubMedGoogle Scholar
  6. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491CrossRefPubMedPubMedCentralGoogle Scholar
  7. Christiaen L, Wagner E, Shi W, Levine M (2009) Electroporation of transgenic DNAs in the sea squirt Ciona. Cold Spring Harbor protocols 2009: pdb. prot5345Google Scholar
  8. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F et al (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cong L, Ran FA, Cox D, Lin S, Barretto R et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  10. Cota CD, Davidson B (2015) Mitotic membrane turnover coordinates differential induction of the heart progenitor lineage. Dev Cell 34:505–519CrossRefPubMedGoogle Scholar
  11. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y et al (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607CrossRefPubMedPubMedCentralGoogle Scholar
  12. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW et al (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fusi, N., I. Smith, J. Doench and J. Listgarten, 2015 In Silico Predictive Modeling of CRISPR/Cas9 guide efficiency. bioRxiv: 021568Google Scholar
  14. Gandhi S, Haeussler M, Razy-Krajka F, Christiaen L, Stolfi A (2017) Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona. Dev Biol 425:8–20Google Scholar
  15. Garneau JE, Dupuis M-È, Villion M, Romero DA, Barrangou R et al (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71CrossRefPubMedGoogle Scholar
  16. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci 109:E2579–E2586CrossRefPubMedPubMedCentralGoogle Scholar
  17. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE et al (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33:510–517CrossRefPubMedPubMedCentralGoogle Scholar
  19. Iaffaldano B, Zhang Y, Cornish K (2016) CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using agrobacterium rhizogenes without selection. Ind Crop Prod 89:356–362CrossRefGoogle Scholar
  20. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA et al (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefPubMedGoogle Scholar
  21. Jinek M, East A, Cheng A, Lin S, Ma E et al (2013) RNA-programmed genome editing in human cells. elife 2:e00471CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E et al (2014) Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343:1247997CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kawai N, Ochiai H, Sakuma T, Yamada L, Sawada H et al (2012) Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases. Develop Growth Differ 54:535–545CrossRefGoogle Scholar
  24. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT et al (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481–485CrossRefPubMedPubMedCentralGoogle Scholar
  25. Long S, Wang Q, Sibley LD (2016) Analysis of noncanonical calcium-dependent protein kinases in toxoplasma gondii by targeted gene deletion using CRISPR/Cas9. Infect Immun 84:1262–1273CrossRefPubMedPubMedCentralGoogle Scholar
  26. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM et al (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31:294–301CrossRefPubMedPubMedCentralGoogle Scholar
  27. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH et al (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods 10:977–979CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mali P, Yang L, Esvelt KM, Aach J, Guell M et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  29. Miller JC, Tan S, Qiao G, Barlow KA, Wang J et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148CrossRefPubMedGoogle Scholar
  30. Moreno-Mateos MA, Vejnar CE, Beaudoin J-D, Fernandez JP, Mis EK et al (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Meth 12:982–988CrossRefGoogle Scholar
  31. Nishiyama A, Fujiwara S (2008) RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryo. Develop Growth Differ 50:521–529CrossRefGoogle Scholar
  32. Nomura T, Sakurai T, Osakabe Y, Osakabe K, Sakakibara H (2016) Efficient and heritable targeted mutagenesis in mosses using the CRISPR/Cas9 system. Plant Cell Physiol 57:2600–2610CrossRefPubMedGoogle Scholar
  33. Nymark M, Sharma AK, Sparstad T, Bones AM, Winge P (2016) A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 6Google Scholar
  34. Orioli A, Pascali C, Quartararo J, Diebel KW, Praz V et al (2011) Widespread occurrence of non-canonical transcription termination by human RNA polymerase III. Nucleic Acids Res 39:5499–5512CrossRefPubMedPubMedCentralGoogle Scholar
  35. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM et al (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 10:973–976CrossRefPubMedPubMedCentralGoogle Scholar
  36. Perry KJ, Henry JQ (2015) CRISPR/Cas9-mediated genome modification in the mollusc, Crepidula Fornicata. Genesis 53:237–244CrossRefPubMedGoogle Scholar
  37. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183CrossRefPubMedPubMedCentralGoogle Scholar
  38. Roy S, Schreiber E (2014) Detecting and quantifying low level gene variants in sanger sequencing traces using the ab1 peak reporter tool. J Biomol Techn JBT 25:S13Google Scholar
  39. Sasaki H, Yoshida K, Hozumi A, Sasakura Y (2014) CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis. Develop Growth Differ 56:499–510CrossRefGoogle Scholar
  40. Satou Y, Shin-i T, Kohara Y, Satoh N, Chiba S (2012) A genomic overview of short genetic variations in a basal chordate, Ciona Intestinalis. BMC Genomics 13:208CrossRefPubMedPubMedCentralGoogle Scholar
  41. Segade F, Cota C, Famiglietti A, Cha A, Davidson B (2016) Fibronectin contributes to notochord intercalation in the invertebrate chordate, Ciona Intestinalis. EvoDevo 7:21CrossRefPubMedPubMedCentralGoogle Scholar
  42. Stolfi A, Gandhi S, Salek F, Christiaen L (2014) Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9. Development 141:4115–4120CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tian S, Jiang L, Gao Q, Zhang J, Zong M et al (2016) Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell Rep:1–8Google Scholar
  44. Tolkin T, Christiaen L (2016) Rewiring of an ancestral Tbx1/10-Ebf-Mrf network for pharyngeal muscle specification in distinct embryonic lineages. Development 143:3852–3862CrossRefPubMedPubMedCentralGoogle Scholar
  45. Treen N, Yoshida K, Sakuma T, Sasaki H, Kawai N et al (2014) Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona. Development 141:481–487CrossRefPubMedGoogle Scholar
  46. Urban A, Neukirchen S, Jaeger K-E (1997) A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR. Nucleic Acids Res 25:2227–2228CrossRefPubMedPubMedCentralGoogle Scholar
  47. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918CrossRefPubMedPubMedCentralGoogle Scholar
  48. Yoshida K, Treen N, Hozumi A, Sakuma T, Yamamoto T et al (2014) Germ cell mutations of the ascidian Ciona Intestinalis with TALE nucleases. Genesis 52:431–439CrossRefPubMedGoogle Scholar
  49. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Center for Developmental Genetics, Department of BiologyNew York UniversityNew YorkUSA
  2. 2.Division of Biology and Biological EngineeringCalifornia Institute of TechnologyPasadenaUSA
  3. 3.School of Biological SciencesGeorgia Institute of TechnologyAtlantaUSA

Personalised recommendations