Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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 13:829–839
Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–573
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712
Beerli RR, Barbas CF (2002) Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 20:135–141
Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764–764
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 155:1479–1491
Christiaen L, Wagner E, Shi W, Levine M (2009) Electroporation of transgenic DNAs in the sea squirt Ciona. Cold Spring Harbor protocols 2009: pdb. prot5345
Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F et al (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761
Cong L, Ran FA, Cox D, Lin S, Barretto R et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Cota CD, Davidson B (2015) Mitotic membrane turnover coordinates differential induction of the heart progenitor lineage. Dev Cell 34:505–519
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 471:602–607
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 34:184
Fusi, N., I. Smith, J. Doench and J. Listgarten, 2015 In Silico Predictive Modeling of CRISPR/Cas9 guide efficiency. bioRxiv: 021568
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 425:8–20
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 468:67–71
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–E2586
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 17:148
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 33:510–517
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 89:356–362
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 337:816–821
Jinek M, East A, Cheng A, Lin S, Ma E et al (2013) RNA-programmed genome editing in human cells. elife 2:e00471
Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E et al (2014) Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343:1247997
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 54:535–545
Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT et al (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481–485
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 84:1262–1273
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 31:294–301
Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH et al (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods 10:977–979
Mali P, Yang L, Esvelt KM, Aach J, Guell M et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Miller JC, Tan S, Qiao G, Barlow KA, Wang J et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148
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 12:982–988
Nishiyama A, Fujiwara S (2008) RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryo. Develop Growth Differ 50:521–529
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 57:2600–2610
Nymark M, Sharma AK, Sparstad T, Bones AM, Winge P (2016) A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 6
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 39:5499–5512
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 10:973–976
Perry KJ, Henry JQ (2015) CRISPR/Cas9-mediated genome modification in the mollusc, Crepidula Fornicata. Genesis 53:237–244
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 152:1173–1183
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:S13
Sasaki H, Yoshida K, Hozumi A, Sasakura Y (2014) CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis. Develop Growth Differ 56:499–510
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 13:208
Segade F, Cota C, Famiglietti A, Cha A, Davidson B (2016) Fibronectin contributes to notochord intercalation in the invertebrate chordate, Ciona Intestinalis. EvoDevo 7:21
Stolfi A, Gandhi S, Salek F, Christiaen L (2014) Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9. Development 141:4115–4120
Tian S, Jiang L, Gao Q, Zhang J, Zong M et al (2016) Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell Rep:1–8
Tolkin T, Christiaen L (2016) Rewiring of an ancestral Tbx1/10-Ebf-Mrf network for pharyngeal muscle specification in distinct embryonic lineages. Development 143:3852–3862
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 141:481–487
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 25:2227–2228
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 153:910–918
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 52:431–439
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 163:759–771
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Nature Singapore Pte Ltd.
About this chapter
Cite this chapter
Gandhi, S., Razy-Krajka, F., Christiaen, L., Stolfi, A. (2018). CRISPR Knockouts in Ciona Embryos. In: Sasakura, Y. (eds) Transgenic Ascidians . Advances in Experimental Medicine and Biology, vol 1029. Springer, Singapore. https://doi.org/10.1007/978-981-10-7545-2_13
Download citation
DOI: https://doi.org/10.1007/978-981-10-7545-2_13
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-10-7544-5
Online ISBN: 978-981-10-7545-2
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)