Hypercompact TnpB and truncated TnpB systems enable efficient genome editing in vitro and in vivo
Ming Wang, Zhaolin Sun, Yue Liu, Pengbin Yin, Chuanyu Liang, Lin Tan, Lei Wei, Yuzhan Wang, Haikuan Yu, Yunfei Zhu, Xiaoxiang Hu, Ning Li, Ran Zhang
Abstract
The bacterial adaptive immune systems CRISPR-Cas9/Cas12 have revolutionized genome editing in eukaryotic cells and in other various organisms 1 . Among the IS200/605 superfamily transposons, the IscB and TnpB proteins are likely progenitors of Cas9 and Cas12, respectively 2 . Recent studies have revealed functional similarities between these putative nucleases and Cas effector proteins, suggesting their programmable RNA-guided endonuclease activities 3 , 4 . Notably, the ISDra2-TnpB protein from Deinococcus radiodurans has demonstrated efficient DNA cleavage adjacent to the 5′-TTGAT transposon-associated motif (TAM) in HEK293T cells 4 . Moreover, with a compact size of only 408 amino acids, which is one-third of the size of the Cas9 protein, the ISDra2-TnpB system holds great promise for genome engineering applications due to its amenability to high-efficiency delivery 5 , 6 . However, certain important questions remain unanswered, such as the feasibility of using this compact ISDra2-TnpB system to generate gene-edited animals and implement gene therapy in vivo. Furthermore, the potential for streamlining and improving the compact ISDra2-TnpB system has not been fully explored. In this study, we present groundbreaking findings on the generation of mutant mice using ISDra2-TnpB and demonstrate its utility in in vivo adeno-associated virus (AAV)-based genome editing. Additionally, we introduce a truncated supermini TnpB editor (< 400 aa) generated by shortening the C-terminal domain (CTD) of TnpB, which also achieves efficient gene editing in mammalian cells and in mice.