Abstract
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very versatile with the ability to simultaneously target multiple genes within a cell (Varshney et al., 2015). We have developed a CRISPR/Cas9 system to delete large genomic fragments (exceeding 30 kb) in Saccharomyces cerevisiae. One application of this technology is to study the effects of large-scale deletions of non-essential genes which may give insight into the function of gene clusters within chromosomes at the molecular level. In this protocol, we describe the general procedures for large fragment deletion in S. cerevisiae using CRISPR/Cas9 including: how to design CRISPR arrays and how to construct Cas9-crRNA expression plasmids as well as how to detect mutations introduced by the system within S. cerevisiae cells.
Keywords: CRISPR/Cas9 system, Large fragment deletion, Saccharomyces cerevisiae
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.
Materials and Reagents
Equipment
Procedure
Notes
In this protocol, the large-scale deletion efficiency was only 10%. The likely reason for this is that the DSB can be repaired via NHEJ, which typically results in small insertions and/or deletions (indels) at the site of the break. While the HDR pathway allows high fidelity and precise editing, the efficiency of large-scale deletion in S. cerevisiae could be improved if the target vector contains the homology arms of target gene.
Recipes
Note: The solvent for the media is ddH2O.
Acknowledgments
This work was supported by the open fund of Key laboratory (No. 3333112) and Bioengineering key discipline of Hebei Province.
References
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