Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function inCiona

Abstract

Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.

Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9

“…Targeted mutagenesis in Ciona was reported using transcription activator-like effectors (TALEs) and zinc-finger nucleases (Kawai et al, 2012;Treen et al, 2014;Yoshida et al, 2014). Here, we report the use of CRISPR/Cas9 to induce site-specific double-stranded breaks (DSBs) in the C. intestinalis genome.…”

Hox-mediated endodermal identity patterns the pharyngeal muscle formation in the chordate pharynx

“…Hox1 and Otx establish the regional identity of the endostyle To explore the role of Hox1 in the endoderm, we carried out tissuespecific knockout of Hox1 using transcription activator-like effector nucleases (TALENs) (Treen et al, 2014). A TALEN pair designed to target the Hox1 locus was expressed in the endodermal tissues under the control of Titf1 regulatory sequence, which expresses downstream genes in the endoderm and a small subset of neural cells (Ristoratore et al, 1999;Sasakura et al, 2012).…”

“…The activity of the constructed TALENs were estimated by previously described methods (Fig. S4) (Treen et al, 2014). Details of the construction of expression vectors and electroporation procedures are described in supplementary Materials and Methods.…”

Engineered Nucleases Lead to Genome Editing Revolution in Rats

“…We applied Platinum TALENs to target the Il2rg gene in rat zygotes, and the mutant-generating effi ciency was much higher than that of other TALENs (Tesson et al 2011 ;Mashimo et al 2013 ) and ZFNs (Mashimo et al 2010b(Mashimo et al , 2012. Platinum TALENs have already been used in other species Treen et al 2014 ;Nakagawa et al 2014 ) and have made a valuable contribution to genome editing research.…”