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
University 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 5183-5195Subject 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