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bioRxiv · 10.1101/2023.03.06.531077

Reprogramming an RNA-guided archaeal endonuclease for genome editing

Xu, Y.·Liu, T.·Wang, J.·Xiong, B.·Liu, L.·Peng, N.
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Abstract

RNA-guided endonucleases developed for genome editing have been identified exclusively from bacteria until now. Recently, the RNA-guided TnpB endonucleases encoded by bacterial IS200/IS605 and IS607 families have been reprogrammed for genome editing. In fact, these insertion sequence families are also widely distributed in archaea. However, whether archaea-derived TnpB could be used for genome editing remains unclear. Here, we found that TnpB derived from Sulfolobus islandicus REY15A (SisTnpB1) was an Mg2+/Mn2+-dependent RNA-guided endonuclease, and it could cleave DNA next to the 5 TTTAA transposon-associated motif (TAM) to generate double-stranded DNA breaks at the 3-end. We also found that SisTnpB1 was active at 37-85{degrees}C and tolerant to variations of TAM. Moreover, SisTnpB1 could be reprogrammed to edit bacterial cells with an editing efficiency of 100% at 37{degrees}C or 45{degrees}C. In summary, our finding expanded the resources of RNA-guided endonucleases for genome editing, especially those derived from archaea, the third domain of life.

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Xu, Y., Liu, T., Wang, J., Xiong, B., Liu, L., Peng, N.. 2023-03-06. Reprogramming an RNA-guided archaeal endonuclease for genome editing. https://doi.org/10.1101/2023.03.06.531077

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