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Rapid production of SaCas9 in plant-based cell-free lysate for activity testing
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  • Andreas Schiermeyer,
  • Pedro Cerda-Bennasser,
  • Thomas Schmelter,
  • Xin Huang,
  • Paul Christou,
  • Stefan Schillberg
Andreas Schiermeyer
Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie Bereich Molekularbiologie

Corresponding Author:[email protected]

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Pedro Cerda-Bennasser
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Thomas Schmelter
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Xin Huang
University of Lleida School of Agricultural and Forestry Engineering
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Paul Christou
University of Lleida School of Agricultural and Forestry Engineering
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Stefan Schillberg
Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie IME
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Abstract

Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with ten different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.
15 Oct 2021Submitted to Biotechnology Journal
18 Oct 2021Submission Checks Completed
18 Oct 2021Assigned to Editor
31 Oct 2021Reviewer(s) Assigned
03 Jan 2022Editorial Decision: Revise Minor
11 Feb 20221st Revision Received
12 Feb 2022Submission Checks Completed
12 Feb 2022Assigned to Editor
14 Feb 2022Reviewer(s) Assigned
18 Mar 2022Editorial Decision: Accept
Jul 2022Published in Biotechnology Journal volume 17 issue 7 on pages 2100564. 10.1002/biot.202100564