Rapid production of SaCas9 in plant-based cell-free lysate for activity testing
BIOTECHNOLOGY JOURNAL(2021)
摘要
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.
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关键词
activity assay, BY-2 lysate, cell-free protein synthesis, CRISPR-Cas, in vitro transcription, translation
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