(2015) Hubbard, Basil P.; Badran, Ahmed; Zuris, John A.; Guilinger, John P.; Davis, Kevin; Chen, Liwei; Tsai, Shengdar Q.; Sander, Jeffry D.; Joung, J. Keith; Liu, David
Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate TALE nucleases with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.
(2015) Davis, Kevin; Pattanayak, Vikram; Thompson, David; Zuris, John A.; Liu, David
Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen (4-HT)-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.
