2026-04-13T13:28:28Zhttps://recercat.cat/oai/request

oai:recercat.cat:10459.1/845192024-12-05T21:30:07Zcom_2072_3622col_2072_479130

00925njm 22002777a 4500 dc Schiermeyer, Andreas author Cerda‑Bennasser, Pedro author Schmelter, Thomas author Huang, Xin author Christou, Paul author Schillberg, Stefan author 2022-12-11T10:47:33Z 2022-12-11T10:47:33Z 2022 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. http://hdl.handle.net/10459.1/84519 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