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.