Sunday, May 24, 2015

Lab Series# 6: A short introduction to TALEN


Fig 1: Gene Editing Tools
Previously I had written a comment piece on the recent ethical debate on use of CRISPR in Human embryo gene editing (Link). Gene editing is not a new phenomenon. Most of the gene editing to date was done using restriction nuclease and only recently have there been improved methodologies.

The first in the series was Zinc finger nuclease (ZFN), then came the CRISPR technology and finally the TALENs. CRISPR is one of the most well known technologies in gene editing and was said method of the year 2011. The same year, TALENs also received equal recognition. However, it went less noticed. With the recent publication of "Embryo gene editing" using CRISPR, showing that it had produced many "off target effects", there is a renewed interest in TALENs which appears to have almost zero off targets.

Table 1: Comparison of gene editing tools
TALEN stands for Transcription activator-like effector nucleases. TALEN is derived from molecules called as TALE (Transcription activator-like effectors), to which a endonuclease enzyme is fused. TALE is produced by a plant pathogen- Xanthomonas via a type III secretion system. Xanthomonas is a member of Proteobacteria known to infect plants. upon a successful infection the TALE binds host genome and modulates its expression of variety of proteins.

Fig 2: TALEN structure. Source
Structurally TALE consists of 18 repeats of 34 amino acids. The repeat varies at amino acids 12 and 13. These variation are called as RVD (Repeat Variable Diresidue). Different RVDs associate preferentially with different nucleotides, with the four most common RVDs (HD, NG, NI, and NN) accounting for each of the four nucleotides (C, T, A, and G, respectively). By fusing the TALE sequence to a Fok1 nulcease, the synthetic compound is now called a TALEN. However, Fok1 works only in a dimerized form and hence TALENs are always designed as pairs binding opposing strands of the DNA to allow dimerization of FokI in a spacer region that is bridging the two TALE binding sites. The treatment of genome with TALEN lead to specific DNA binding and subsequent DSB (Double strand break) which the genome repairs through a NHEJ or HDR.

The current concerns in use of these high throughput gene editing tools is the off target effects. I'm not aware of any reports of TALEN off target effects or toxicity, the view is that not enough studies has been done to assert the claims. However, the feasibility of laboratory handling is much tough in comparison to CRISPR and hence they still are not the favorites.
Oost, J. (2013). New Tool for Genome Surgery Science, 339 (6121), 768-770 DOI: 10.1126/science.1234726

Gaj T, Gersbach CA, & Barbas CF 3rd (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in biotechnology, 31 (7), 397-405 PMID: 23664777

Bogdanove AJ, & Voytas DF (2011). TAL effectors: Customizable proteins for DNA targeting. Science, 333 (6051), 1843-6 PMID: 21960622

Kim H, & Kim JS. (2014) A guide to genome engineering with programmable nucleases. Nature reviews. Genetics, 15(5), 321-34. PMID: 24690881