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In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells.
Journal article

In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells.

  • Savić N The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
  • Ringnalda FC The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
  • Berk C Institute for Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
  • Bargsten K Department of Biochemistry, University of Zurich, Zurich, Switzerland.
  • Hall J Institute for Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
  • Jinek M Department of Biochemistry, University of Zurich, Zurich, Switzerland.
  • Schwank G The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
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  • 2019-01-25
Published in:
  • Bio-protocol. - 2019
CRISPR-Cas9
Enhanced correction
Genome editing
Homology-directed repair
Precise editing
Template linkage
English The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells.
Language
  • English
Open access status
hybrid
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Persistent URL
https://sonar.ch/global/documents/36863
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