Enabling large-scale genome editing by reducing DNA nicking

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-s...

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Bibliographic Details
Published inbioRxiv
Main Authors Smith, Cory J, Castanon, Oscar, Said, Khaled, Volf, Verena, Khoshakhlagh, Parastoo, Hornick, Amanda, Ferreira, Raphael, Chun-Ting, Wu, Güell, Marc, Garg, Shilpa, Myllykallio, Hannu, Church, George M
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 04.04.2019
Subjects
Cell death
Cell survival
Copy number
DNA damage
Genome editing
Genomes
Mutation
Nicking endonuclease
Pluripotency
Stem cells
Toxicity
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Summary: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. Footnotes * Figures aesthetically improved and minor typos corrected.
DOI:10.1101/574020