Enabling large-scale genome editing by reducing DNA nicking

Smith, C. J.; Castanon, O.; Said, K.; Volf, V.; Khoshakhlagh, P.; Hornick, A.; Ferreira, R.; Wu, C.-T.; Güell, M.; Garg, S.; Myllykallio, H.; Church, G. M.

doi:10.1101/574020

bioRxiv · 10.1101/574020

Enabling large-scale genome editing by reducing DNA nicking

Smith, C. J.·Castanon, O.·Said, K.·Volf, V.·Khoshakhlagh, P.·Hornick, A.·Ferreira, R.·Wu, C.-T.·Güell, M.·Garg, S.·Myllykallio, H.·Church, G. M.

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Abstract

To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements - ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.\n\nOne Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

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Smith, C. J., Castanon, O., Said, K., Volf, V., Khoshakhlagh, P., Hornick, A., Ferreira, R., Wu, C.-T., Güell, M., Garg, S., Myllykallio, H., Church, G. M.. 2019-03-15. Enabling large-scale genome editing by reducing DNA nicking. https://doi.org/10.1101/574020

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