CRISPR Knockouts in Ciona Embryos
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.
KeywordsGenome 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.
- 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
- Fusi, N., I. Smith, J. Doench and J. Listgarten, 2015 In Silico Predictive Modeling of CRISPR/Cas9 guide efficiency. bioRxiv: 021568Google Scholar
- 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
- 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
- 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
- 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