Engineering Designer Transcription Activator‐‐Like Effector Nucleases (TALENs) by REAL or REAL‐Fast Assembly

Abstract

Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe a detailed protocol for the serial, hierarchical assembly of TALENs that requires neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. This restriction enzyme and ligation (REAL) protocol can be practiced using a small plasmid library and user-friendly, web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. All plasmids required to perform the REAL assembly method are publicly available through the Addgene plasmid distribution service (http://www.addgene.org/talengineering). Alternatively, to decrease the time and effort required, users can practice REAL using a library of plasmids encoding pre-assembled TALE repeats. With the platform of reagents, protocols, and software we describe, researchers can easily engineer multiple TALENs within two weeks or less using standard cloning techniques.

A Micropeptide Encoded by a Putative Long Noncoding RNA Regulates Muscle Performance

“…A unique TALEN pair specific for exon 1 of the MLN locus was designed and constructed using the REAL assembly method (Reyon et al, 2012). A loss of function allele was created using a donor plasmid to insert a red fluorescent reporter (tdTomato) followed by a triple polyadenylation cassette into exon 1 in the MLN locus (Figure 6A).…”

“…A unique TALEN pair specific for the MLN locus was designed using the ZiFiT Targeter Program (http://zifit.partners.org/ZiFiT/Introduction.aspx) and constructed using the REAL assembly kit (Addgene) (Reyon et al, 2012). A donor vector containing the tdTomato reporter and triple polyadenylation sequences was constructed by incorporating short 5’ and 3’ homology arms specific to the MLN locus.…”

Albumin-deficient mouse models for studying metabolism of human albumin and pharmacokinetics of albumin-based drugs

“…methodology. 39,40 The TALEN pair targeted 5'-TTTCCTGCAACACAAAGA tgacaaccccagcctg CCAC-CATTTGAAAGGCCA-3 0 (TALEN recognition sequences capitalized, intervening sequence in lowercase). Capped and tailed mRNA encoding each TALEN was prepared using the AmpliCap-Max TM T7 High Yield Message Maker Kit (Cellscript, Cat.…”

Targeted chromosomal deletions and inversions in zebrafish

“…We changed the overall architecture of the final backbone of the Golden Gate System, since the original TALEN constructs contained longer N-and C-terminal elements than those used in zebrafish Sander et al 2011). Since pJDS vectors (Sander et al 2011) from the Joung Laboratory REAL Assembly TALEN Kit (Addgene) (Reyon et al 2012a) were highly active in zebrafish (Sander et al 2011), we used the architecture from pJDS vectors for the incorporation of the Golden Gate assembled TALE arrays. To make pJDS vectors compatible with the Voytas laboratory Golden Gate Kit, the overhangs generated by Esp3I cleavage sites in the pJDS vectors were adjusted to GGGA/CTAT to ensure proper complementarity between intermediate vectors and the final vector.…”