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.