Balance between DNA-binding affinity and specificity enables selective recognition of longer target sequences in vivo

Although genome-editing enzymes such as TALEN and CRISPR/Cas9 are being widely used, they have an essential limitation in that their relatively high-molecular weight makes them difficult to be delivered to cells. To develop a novel genome-editing enzyme with a smaller molecular weight, we focused on the engrailed homeodomain (EHD). We designed and constructed proteins composed of two EHDs connected by a linker to increase sequence specificity. In bacterial one-hybrid assays and electrophoresis mobility shift assay analyses, the created proteins exhibited good affinity for DNA sequences consisting of two tandemly aligned EHD target sequences. However, they also bound to individual EHD targets. To avoid binding to single target sites, we introduced amino acid mutations to reduce the protein-DNA affinity of each EHD monomer and successfully created a small protein with high specificity for tandem EHD target sequences.