Megabase-scale human genome rearrangement with programmable bridge recombinases
Nicholas Thomas Perrya b c d e, Liam J. Bartiea b c d e, Dhruva Katrekara d, Gabriel A. Gonzaleza d, Matthew G. Durranta d, James J. Paia d, Alison Fantona b c d e, Juliana Q. Martinsa d f, Masahiro Hiraizumig h, Chiara Ricci-Tama d, Hiroshi Nishimasug i j k l, Silvana Konermanna d m✉, Patrick D. Hsua b d n✉
- aArc Institute, Palo Alto, CA, USA
- bDepartment of Bioengineering, University of California, Berkeley, Berkeley, CA, USA
- cUniversity of California, Berkeley - University of California, San Francisco, Graduate Program in Bioengineering, Berkeley, CA, USA
- dArc Institute, 3181 Porter Drive, Palo Alto, CA, USA
- eUniversity of California, Berkeley - University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA
- fDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA
- gDepartment of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
- hDepartment of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan
- iInamori Research Institute for Science, Shimogyo-ku, Kyoto, Japan
- jStructural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo, Japan
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Abstract
Bridge recombinases are naturally occurring RNA-guided DNA recombinases that we previously demonstrated can programmably insert, excise, and invert DNA in vitro and in Escherichia coli . In this study, we report the discovery and engineering of the bridge recombinase ortholog ISCro4 for universal rearrangements of the human genome. We defined strategies for the optimal application of bridge systems, leveraging mechanistic insights to improve their targeting specificity. Through rational engineering of the ISCro4 bridge RNA and deep mutational scanning of its recombinase, we achieved up to 20% insertion efficiency into the human genome and genome-wide specificity as high as 82%. We further demonstrated intrachromosomal inversion and excision, mobilizing up to 0.93 megabases of DNA. Lastly, we provided proof of concept for plasmid-based excision of disease-relevant gene regulatory regions or repeat expansions.