Enabling large-scale genome editing at repetitive elements by reducing DNA nicking

Smith, Cory J.; Castanon, Oscar; Said, Khaled; Volf, Verena; Khoshakhlagh, Parastoo; Hornick, Amanda; Ferreira, Raphael; Wu, Chun-Ting; Güell Cargol, Marc, 1982-; Garg, Shilpa; Ng, Alex H. M.; Myllykallio, Hannu; Church, George M.

doi:http://dx.doi.org/10.1093/nar/gkaa239

Enabling large-scale genome editing at repetitive elements by reducing DNA nicking

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Citation

Smith CJ, Castanon O, Said K, Volf V, Khoshakhlagh P, Hornick A, Ferreira R, Wu CT, Güell M, Garg S, Ng AHM, Myllykallio H, Church GM. Enabling large-scale genome editing at repetitive elements by reducing DNA nicking. Nucleic Acids Res. 2020; 48(9):5183-95. DOI: 10.1093/nar/gkaa239

Abstract

To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of 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 and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 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.