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 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.

ALiCE®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

“…We have shown that BYL can produce functional proteins as diverse as enzymes, multi-pass transmembrane GPCRs, small, soluble growth factors and immunologically-active viral antigens. Moreover, functional expression of various biosynthetic enzymes, Cas9 nuclease and the antibody M12 have previously been reported from BYL 10,40,41 . Protein function is intrinsically linked to correct folding but further molecular characterisation was warranted to prove that the microsomes of this eukaryotic CFPS system are able to produce recombinant proteins at the same quality as a cell and with batch-to-batch consistency.…”

Editorial: CRISPR and alternative approaches

“…Schiermeyer et al. [ 11 ] report the establishment of a robust platform for the verification of selected gRNAs and rapid production of SaCas9 in cell‐free lysate from tobacco ( Nicotiana tabacum L.) BY‐2 cells. The in vitro assay can be completed before in planta experiments.…”