Effective genome editing with an enhanced ISDra2 TnpB system and deep learning-predicted ωRNAs.

Journal: Nature methods
PMID:

Abstract

Transposon (IS200/IS605)-encoded TnpB proteins are predecessors of class 2 type V CRISPR effectors and have emerged as one of the most compact genome editors identified thus far. Here, we optimized the design of Deinococcus radiodurans (ISDra2) TnpB for application in mammalian cells (TnpBmax), leading to an average 4.4-fold improvement in editing. In addition, we developed variants mutated at position K76 that recognize alternative target-adjacent motifs (TAMs), expanding the targeting range of ISDra2 TnpB. We further generated an extensive dataset on TnpBmax editing efficiencies at 10,211 target sites. This enabled us to delineate rules for on-target and off-target editing and to devise a deep learning model, termed TnpB editing efficiency predictor (TEEP; https://www.tnpb.app ), capable of predicting ISDra2 TnpB guiding RNA (ωRNA) activity with high performance (r > 0.8). Employing TEEP, we achieved editing efficiencies up to 75.3% in the murine liver and 65.9% in the murine brain after adeno-associated virus (AAV) vector delivery of TnpBmax. Overall, the set of tools presented in this study facilitates the application of TnpB as an ultracompact programmable endonuclease in research and therapeutics.

Authors

  • Kim Fabiano Marquart
    Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
  • Nicolas Mathis
    Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
  • Amina Mollaysa
    Department of Quantitative Biomedicine, University of Zurich, Zurich, Switzerland.
  • Saphira Müller
    Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland.
  • Lucas Kissling
    Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
  • Tanja Rothgangl
    Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland.
  • Lukas Schmidheini
    Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
  • Péter István Kulcsár
    Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland.
  • Ahmed Allam
    Department of Quantitative Biomedicine, University of Zurich, Zurich, Switzerland.
  • Masako M Kaufmann
    Institute for Transfusion Medicine and Gene Therapy, Medical Center, University of Freiburg, Freiburg, Germany.
  • Mai Matsushita
    Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.
  • Tatjana Haenggi
    Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland.
  • Toni Cathomen
    Institute for Transfusion Medicine and Gene Therapy, Medical Center, University of Freiburg, Freiburg, Germany.
  • Manfred Kopf
    Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.
  • Michael Krauthammer
    Yale School of Medicine, New Haven, CT.
  • Gerald Schwank
    Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland. schwank@pharma.uzh.ch.