TY - JOUR
T1 - Divalent cations promote TALE DNA-binding specificity
AU - Cuculis, Luke
AU - Zhao, Chuankai
AU - Abil, Zhanar
AU - Zhao, Huimin
AU - Shukla, Diwakar
AU - Schroeder, Charles M.
N1 - Funding Information:
D.S. and C.Z. acknowledge the support from the Blue Waters sustained-petascale computing project, which is supported by the National Science Foundation (awards OCI-0725070 and ACI-1238993) and the state of Illinois.
Publisher Copyright:
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2020/2/20
Y1 - 2020/2/20
N2 - Recent advances in gene editing have been enabled by programmable nucleases such as transcription activator-like effector nucleases (TALENs) and CRISPR–Cas9. However, several open questions remain regarding the molecular machinery in these systems, including fundamental search and binding behavior as well as role of off-target binding and specificity. In order to achieve efficient and specific cleavage at target sites, a high degree of target site discrimination must be demonstrated for gene editing applications. In this work, we studied the binding affinity and specificity for a series of TALE proteins under a variety of solution conditions using in vitro fluorescence methods and molecular dynamics (MD) simulations. Remarkably, we identified that TALEs demonstrate high sequence specificity only upon addition of small amounts of certain divalent cations (Mg2+, Ca2+). However, under purely monovalent salt conditions (K+, Na+), TALEs bind to specific and non-specific DNA with nearly equal affinity. Divalent cations preferentially bind to DNA over monovalent cations, which attenuates non-specific interactions between TALEs and DNA and further stabilizes specific interactions. Overall, these results uncover new mechanistic insights into the binding action of TALEs and further provide potential avenues for engineering and application of TALE- or TALEN-based systems for genome editing and regulation.
AB - Recent advances in gene editing have been enabled by programmable nucleases such as transcription activator-like effector nucleases (TALENs) and CRISPR–Cas9. However, several open questions remain regarding the molecular machinery in these systems, including fundamental search and binding behavior as well as role of off-target binding and specificity. In order to achieve efficient and specific cleavage at target sites, a high degree of target site discrimination must be demonstrated for gene editing applications. In this work, we studied the binding affinity and specificity for a series of TALE proteins under a variety of solution conditions using in vitro fluorescence methods and molecular dynamics (MD) simulations. Remarkably, we identified that TALEs demonstrate high sequence specificity only upon addition of small amounts of certain divalent cations (Mg2+, Ca2+). However, under purely monovalent salt conditions (K+, Na+), TALEs bind to specific and non-specific DNA with nearly equal affinity. Divalent cations preferentially bind to DNA over monovalent cations, which attenuates non-specific interactions between TALEs and DNA and further stabilizes specific interactions. Overall, these results uncover new mechanistic insights into the binding action of TALEs and further provide potential avenues for engineering and application of TALE- or TALEN-based systems for genome editing and regulation.
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U2 - 10.1093/NAR/GKZ1174
DO - 10.1093/NAR/GKZ1174
M3 - Article
C2 - 31863586
AN - SCOPUS:85082144587
SN - 0305-1048
VL - 48
SP - 1406
EP - 1422
JO - Nucleic acids research
JF - Nucleic acids research
IS - 3
ER -