4 Discussion
Our experimental data demonstrate the rapid and efficient production of
rSaCas9 in the cell-free BYL transcription/translation system (Figure 1)
within a time frame of one to two days. Although the molecular mass of
rSaCas9 is approximately four times higher than that of the
well-established positive control eYFP, its production is clearly
visible within crude BYL after gel electrophoresis and protein staining.
Enrichment of rSaCas9 and removal of tobacco host proteins was achieved
with a single affinity purification step using a C-terminally fused
Strep-tag II (Figure 3). The entire procedure of enzyme production and
purification is rather simple and only requires a moderate amount of
hands-on time. Importantly, the preparation of cell-free lysates and
protein biosynthesis can be decoupled. After lysate preparation,
ready-to-use reaction samples can be stored at -80 °C for up to one
year. If necessary, template DNA (plasmid or PCR fragment) carrying the
expression cassette encoding the target protein can be added to start
protein synthesis. Thus, the cell-free platform provides a flexible,
on-demand tool to provide sufficient amounts of Cas9 nuclease for
various assays without elaborate and cost-intensive infrastructure for
cell-based protein production systems.
With an estimated yield of 0.1 mg of purified rSaCas9, the enzyme amount
was sufficient to conduct several hundred in vitro cleavage
assays under the conditions used here. However, there is potential to
optimize the process and improve enzyme yields further. A fraction of
rSaCas9 is insoluble and appears in the pellet fraction during the
purification process (Figure 3). To increase the solubility of rSaCas9,
the process parameters of the IVT reaction, such as the incubation
temperature, could be adjusted. The affinity purification process
resulted in highly pure rSaCas9 (Figure 3), but the enzyme was not
captured quantitatively on the matrix, as an rSaCas9 band is visible on
the gel from the flow-through and wash fractions. To improve the
enzyme–matrix interaction, the Strep-tag II might be moved to the other
protein terminus, or a Twin-Strep-tag might be used instead [15].
The programmable nuclease activity of purified rSaCas9 was demonstrated
by the addition of various sgRNAs that directed the cleavage of ten
different target sites within the maize CCD8 promoter sequence (Figure
4). Therefore, we confirmed that this cell-free codon-optimized version
of rSaCas9 with additional elements for nuclear targeting is active on
selected target sites and can be used for stable transformation
experiments in maize to induce mutations in the CCD8 promoter sequence.
The verification of enzyme activity on selected target sites was the
primary aim of this study. However, given the simplicity, robustness and
speed of the cell-free BYL system, we envisage its use for a variety of
other applications in genome engineering projects. These include
engineering Cas9 nucleases for higher versatility, e.g., by engineering
versions that are less dependent on a PAM sequence [16] [17] for
their activity. As in vitro transcription/translation platforms
are easily adaptable to high-throughput formats [18, 19], the
parallel testing of multiple Cas9 enzymes with altered properties could
be achieved. In addition to testing mutant Cas9 variants, the system
might also be useful to assess the properties of Cas9 fusion proteins
for different purposes, e.g., base editing [20], epigenetic
modification [21], or transcriptional activation/repression
[22]. Once a robust Cas9 nuclease has been identified for a
particular purpose, the BYL system can be used to produce RNPs for
subsequent DNA-free delivery to the appropriate target cell/tissue.