Rapid production of SaCas9 in plant-based cell-free lysate for activity testing

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 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.