Enabling large-scale genome editing by reducing DNA nicking

bioRxiv preprint first posted online Mar. 15, 2019; doi: http://dx.doi.org/10.1101/574020. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Authors
Cory J. Smith, Oscar Castanon, Khaled Said, Verena Volf, Parastoo Khoshakhlagh, Amanda Hornick, Raphael Ferreira, Chun-Ting Wu, Marc Güell6, Shilpa Garg1, Hannu Myllykallio, George M. Church.
03-27-2019 HSW1057
12:00pm
PST
Categories
RNA Modification & Editing
Speaker
Stacey Phan
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 (dBEs) 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 5 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.