2026-04-13T02:33:27Zhttps://recercat.cat/oai/requestoai:recercat.cat:10459.1/845192024-12-05T21:30:07Zcom_2072_3622col_2072_479130
RECERCAT
author
Schiermeyer, Andreas
author
Cerda‑Bennasser, Pedro
author
Schmelter, Thomas
author
Huang, Xin
author
Christou, Paul
author
Schillberg, Stefan
2024-12-05T21:30:07Z
2024-12-05T21:30:07Z
2022-12-11T10:47:33Z2022-12-11T10:47:33Z2022
http://hdl.handle.net/10459.1/84519
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 DNAfree
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-kDaCas9 nuclease from Staphylococcus aureus (SaCas9) was
produced and subsequently purified via affinity chromatography. The purified apoenzyme
was supplemented with 10 different sgRNAs, and the nuclease activity was confirmed
by the linearization of plasmid DNA containing cloned DNA target sequences.We are grateful to Dr. Matthias Buntru (Fraunhofer IME) for providing BY-2 cell lysate and valuable advice on in vitro transcription/ translation. Financial support for this research has been provided by the German Federal Ministry of Education and Research (BMBF, grant number 031B0813) and by the Spanish Ministry of Economy and Competitiveness (MINECO), Spain (RTI2018-097613-BI00; PGC2018-097655-B-I00); PROSTRIG, ERA-NET Cofund SusCrop (Grant No 771134). AS, PC, and SS conceived the study. AS, PCB, XH, and TS conducted experiments and collected data. AS wrote the manuscript. All authors interpreted the data and proofread and approved the manuscript.
cc-by (c) Andreas Schiermeyer et. al., 2022. info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/4.0/
Activity assay
BY-2 lysate
Cell-free protein synthesis
CRISPR-Cas
In vitro transcription/ translation
Rapid production of SaCas9 in plant-based cell-free lysate for activity testing
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion