Abstract
The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that facilitate high expression of sgRNA and improved CRISPR-Cas9 function. We provide selection criteria of Pol III promoters, efficient promoter construction methods, and a sample screening technique to test the efficiency of the hybrid promoters. A hybrid promoter system developed for Yarrowia lipolytica will serve as a model.
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