(via T. Lahaye, thx)
Mali et al (2013)
As a genome editing tool TAL effector–FokI dimers are usually used, and for genome regulation TAL effector–VP64 fusions have been shown to be highly effective. We used the latter as ... this format also reveals the specificity profile of individual TAL-effectors. Examining the TAL effector off-targeting data ... reveals that 18-mer TAL effectors can potentially tolerate 1 or 2 mutations in their target sequences, but fail to activate a large majority of three-base mismatch variants in their targets. They are also particularly sensitive to mismatches nearer
the 5ʹ end of their target sequences. Notably, certain mutations in the middle of the target lead to greater TAL effector activity, an aspect that needs further evaluation. We confirmed a subset of the above results through targeted experiments in a nuclease assay ... We also observed that shorter TAL effectors (14-mer and 10-mer) are progressively less tolerant of mismatches but also reduced in activity by an order of magnitude...
To decouple the role of individual repeat-variable di-residues (RVDs), we confirmed that choice of RVDs does contribute to base specificity but TAL effector specificity is also a function of the binding energy of the protein as a whole ... our data imply that engineering shorter TAL effectors or TAL effectors bearing a judicious composition of high- and low-affinity monomers can potentially yield higher specificity in genome engineering applications, and the requirement for FokI dimerization in nuclease applications enables a further dramatic reduction in off-target effects especially when using the shorter TAL effectors.