Background

The CRISPR/Cas9 system is a rapid, efficient, low-cost, and versatile method for genome editing that can be applied in the fields of biology, agriculture, and medicine. To date, several protocols have been reported on how to make large-scale deletions within genomes. Each of these methods contains its own unique characteristics and advantages. The recently developed CRISPR/Cas9 system for excising large stretches of chromosomes has potential advantages over other methods such as the Latour system (Hirashima et al., 2006). The CRISPR/Cas9 system requires two components: (1) the Cas9 endonuclease for DNA cleavage and (2) a variable guide RNA (gRNA) that directs the Cas9 enzyme in a DNA sequence-specific manner (Cong et al., 2013). When Cas9 is targeted to a genomic locus by a gRNA, Cas9 initiates a DSB. The cell will respond to the DSB by repairing the damage via one of two major pathways: high-fidelity homology-dependent repair or error-prone non-homologous end joining (NHEJ).

The CRISPR/Cas9 system described here requires four components: Cas9 endonuclease, CRISPR array, trans-activating crRNA (tracrRNA), and RNase III (this activity is present in the host cell). The CRISPR array is a genomic locus from which pre-crRNAs are transcribed. In this system, the CRISPR array, engineered on the pCRCT plasmid, expresses multiple spacers flanked by direct repeats driven by a single promoter. Cas9 cannot be targeted by crRNA alone, it requires a crRNA-tracrRNA duplex to target it to a specific site within the genome. Two DNA oligonucleotides that encode for spacer sequences interspaced by a direct repeat (DR) were directly synthesized. The formed dsDNA encoding the crRNA was cloned into a Cas9 expression vector. Once the desired plasmid is constructed, transform S. cerevisiae with it and screen the transformants to obtain mutants with large genomic fragment deletions. In this protocol, the deletion efficiency (10%) is lower than described for deletion of genomic fragments using CRISPR/Cas9 in rice (Zhou et al., 2014). There are two possible reasons. The first one is that the genome may be repaired more rapidly in S. cerevisiae in comparison with that in rice. Another one is a stronger selection pressure used for screening rice transformants. In rice, the selection marker for transformants is hygromycin B, while in yeast Uracil as a selection marker is employed. The stronger selection pressure possibly increase the plasmid copy numbers in rice cells.