Enabling large-scale genome editing at repetitive elements by reducing DNA nicking. Issue 9 (21st April 2020)

• Record Type: Journal Article
• Authors: Smith, Cory J; Castanon, Oscar; Said, Khaled; Volf, Verena; Khoshakhlagh, Parastoo; Hornick, Amanda; Ferreira, Raphael; Wu, Chun-Ting; Güell, Marc; Garg, Shilpa; Ng, Alex H M; Myllykallio, Hannu; Church, George M
• Is Part Of: Nucleic acids research. Volume 48:Issue 9(2020)
• Page Start: 5183
• Page End: 5195
• Publication Date: 2020-04-21
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkaa239
• Languages: English
• ISSNs: 0305-1048

Abstract

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.