Enabling large-scale genome editing at repetitive elements by reducing DNA nicking
Journal article, 2020

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.

Author

Cory J. Smith

Harvard Medical School

Harvard University

Oscar Castanon

École polytechnique

Harvard Medical School

Harvard University

Khaled Said

Harvard University

Harvard Medical School

Verena Volf

Harvard Medical School

Harvard School of Engineering and Applied Sciences

Harvard University

Parastoo Khoshakhlagh

Harvard Medical School

Harvard University

Amanda Hornick

Harvard Medical School

Harvard University

Raphael Ferreira

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Chun Ting Wu

Harvard University

Harvard Medical School

Marc Güell

Universitat Pompeu Fabra

Shilpa Garg

Harvard Medical School

Alex H.M. Ng

Harvard Medical School

Harvard University

Hannu Myllykallio

École polytechnique

George M. Church

Harvard University

Harvard Medical School

Nucleic Acids Research

0305-1048 (ISSN) 1362-4962 (eISSN)

Vol. 48 9 5183-5195

Subject Categories

Cell Biology

Cell and Molecular Biology

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

DOI

10.1093/nar/gkaa239

PubMed

32315033

More information

Latest update

9/23/2020