1 Introduction
The precise modification of chromosomal DNA sequences by genome editing
offers a wide range of applications in basic research and applied
biotechnology. The CRISPR-Cas system has gained widespread acceptance
due to its easy implementation. In contrast to other programmable
nucleases (e.g., ZFN or TALEN), the CRISPR-Cas system does not require
complex protein engineering, as the sequence specificity is brought
about by a guide RNA that is easy to design and modify. To deliver the
components into the target tissue or cell, various approaches have been
used: delivery of DNA constructs coding for Cas9 and sgRNA, delivery of
the corresponding transcripts, or delivery of preassembled RNPs [1].
The delivery of DNA constructs usually leads to the integration of the
expression cassette into the host genome and therefore a constant
presence of the Cas9 nuclease and the sgRNA molecules. However, this may
result in undesired effects, such as gene inactivation or rearrangements
at the integration site. The delivery of RNA transcripts provides only
transient expression of Cas9, as RNA molecules usually have a short
half-life. In contrast, preassembled RNPs consisting of single guide
RNAs (sgRNA)s and recombinant Cas9 nucleases are not integrated into the
host genome, are more stable than transcripts, and usually lead to
higher editing efficiencies [2]. To date, the production of
recombinant Cas9 nuclease relies on E. coli strains as production
hosts followed by purification of the enzyme [3].
The most commonly used Cas9 nucleases are derived fromStreptococcus pyogenes (SpCas9) and Staphylococcusaureus (SaCas9). Although the two nucleases catalyze the same
reaction, their properties are different. Both enzymes introduce a DNA
double strand break through the action of the two nuclease domains RuvC
and HNH. However, the SaCas9 enzyme, consisting of 1053 amino acids, is
significantly smaller than the SpCas9 enzyme, with 1368 amino acids
[4]. Both nucleases cleave in close proximity to the protospacer
adjacent motif (PAM), which is defined as 5’-NGG for SpCas9 [5] and
5’ NNGRRT for SaCas9 [6], although the molecular mechanisms of
cleavage vary between the two enzymes [7, 8, 9].
Here, we demonstrate that tobacco BY-2 cell-free lysate (BYL) is a
powerful platform to provide recombinant SaCas9 at sufficient quantity
and quality to verify its nuclease activity on specified DNA target
sequences. In particular, we used this assay to validate the nuclease
activity of rSaCas9 on target sites within the promoter region of the
maize gene coding for carotenoid cleavage dioxygenase 8 (CCD8). Thus,
the results of this rapid in vitro assay provided valuable data
before embarking on in planta experiments.