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

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.

SUBMITTER: Smith CJ

PROVIDER: S-EPMC7229841 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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

Smith Cory J CJ Castanon Oscar O Said Khaled K Volf Verena V Khoshakhlagh Parastoo P Hornick Amanda A Ferreira Raphael R Wu Chun-Ting CT Güell Marc M Garg Shilpa S Ng Alex H M AHM Myllykallio Hannu H Church George M GM

Nucleic acids research 20200501 9

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 ...[more]

PMID: 32315033

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