Transcription activator-like effector nucleases (TALENs) have rapidly emerged as a powerful genome editing tool. The site-specific DNA double-strand breaks generated by TALENs in the human chromosome can induce homologous recombination or non-homologous end joining, resulting in desired genetic modifications. In this study, we report the development of a TALEN variant, SunnyTALEN, with >2.5-fold improved genome editing efficacy in human cells. The corresponding scaffold increases the rate of genetic modification at all the 13 tested loci of human genome and is compatible with heterodimer TALEN architectures. This enhanced and high-efficiency TALEN variant represents a novel second-generation TALEN system and has great potential for biological and therapeutic applications. Biotechnol. Bioeng. 2014;111: 683–691. © 2013 Wiley Periodicals, Inc.