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Publications

Journal Article Research Support, Non-U.S. Gov't Ppublish 21 citations

PubMed: 38459694 DOI: 10.1016/j.ymthe.2024.03.006

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.

Mol Ther 32(5):1298-1310

Authors: Julia Klermund, Manuel Rhiel, Thomas Kocher, Kay Ole Chmielewski, Johannes Bischof, Geoffroy Andrieux, Melina El Gaz, Stefan Hainzl, Melanie Boerries, Tatjana I Cornu, Ulrich Koller, Toni Cathomen

Affiliations

  • Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, 79106 Freiburg, Germany.
  • EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria.
  • Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, 79106 Freiburg, Germany; PhD Program, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
  • Institute of Medical Bioinformatics and Systems Medicine, Medical Center - University of Freiburg, 79110 Freiburg, Germany; Faculty of Medicine, University of Freiburg, 79110 Freiburg, Germany.
  • Institute of Medical Bioinformatics and Systems Medicine, Medical Center - University of Freiburg, 79110 Freiburg, Germany; Faculty of Medicine, University of Freiburg, 79110 Freiburg, Germany; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, 79106 Freiburg, Germany.
  • Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, 79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, 79110 Freiburg, Germany.
  • Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, 79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, 79110 Freiburg, Germany.

Abstract

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.

MeSH

Chromosome Aberrations, Genetic Therapy, Clustered Regularly Interspaced Short Palindromic Repeats, Cells, Genes, Genome

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