TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain

Abstract

DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.

Tapping natural reservoirs of homing endonucleases for targeted gene modification

“…S everal types of highly specific DNA recognition and cleavage enzymes, including homing endonucleases (HEs), zinc finger nucleases (ZFNs), and transcription activator-like (TAL) effector nucleases, are being developed for targeted gene modification, ranging from gene disruption to corrective gene therapy (1)(2)(3)(4)(5). Regardless of the identity of the protein scaffold, site-specific gene modification generally requires the creation of individually tailored site-specific endonucleases that generate double-strand breaks (DSBs) at unique chromosomal targets.…”

Are genes switched on when they kiss?

“…4A). [35][36][37][38] Fusion of the FokI endonuclease to a ZF or TALE DNA binding region, or the CRISPR-associated protein Cas9, enables these tools to induce site-specific double strand breaks (DSB). [37][38][39] Two features of FokI have made it an ideal enzyme to be used as a component of a site-specific nuclease.…”

“…[35][36][37][38] Fusion of the FokI endonuclease to a ZF or TALE DNA binding region, or the CRISPR-associated protein Cas9, enables these tools to induce site-specific double strand breaks (DSB). [37][38][39] Two features of FokI have made it an ideal enzyme to be used as a component of a site-specific nuclease. First, the wellcharacterized modular nature of the enzyme [40][41][42][43] has allowed just its catalytic domain to be fused with customizable DNA binding proteins such as zinc fingers and TALEs.…”

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

“…These TAL effectors in conjunction with the Fok1 nuclease (TALENs) have been shown to function similar to ZFNs [50,51] . The TALEN technology provides scientists with an increase in modularity allowing for the creation of many variants at little time and cost.…”