Abstract
Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.
Highlights
The ability of proteins to recognize DNA in a sequencedependent manner is central to life
We provide the first example of a transcription activator-like effector (TALE) recombinase (TALER)
We investigated the effect core sequence length has on recombination by evaluating whether DNA targets containing 14 (Avr-14G), 26 (Avr-26G) and 32-bp (Avr-32G) core sites could be recombined by selected TALERs
Summary
The ability of proteins to recognize DNA in a sequencedependent manner is central to life. A variety of protein domains have evolved to provide sequence-specific DNA recognition. DNA recognition by a select few of these domains is the foundation for a wide variety of biotechnological applications. C2H2 zinc-finger proteins (ZFPs) were among the first DNA-binding proteins to be engineered to recognize user-defined DNA sequences and have been used with varying degrees of success for many applications, including transcriptional regulation, genome engineering and epigenetic modification [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20]. Despite the advances and promise of ZFP technology, construction of specific, high-affinity ZFPs for certain sequences remains difficult and in select cases requires the use of time-consuming and labor-intensive selection systems not readily adopted by non-specialty laboratories [21]
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