Strong expression of Cas9 under a new 3′-truncated TEF1α promoter enhances genome editing in Yarrowia lipolytica

Abstract

The non-conventional yeast Yarrowia lipolytica is gaining interest in biotechnology as a workhorse for the production of proteins, lipids and other biomolecules. Site-specific genome editing is however limited in this yeast. Although, this was much improved by the recent adaptation of a CRISPR-Cas9 genome editing protocol for Y. lipolytica based on a tRNA-sgRNA fusion, yet, in the latter protocol, Cas9 is under the control of a synthetic hybrid promoter, pUAS1B8-TEF(136) that is associated with some drawbacks. This hybrid promoter contains tandem repeats are suggested to cause in vivo and in vitro inconveniences like polymerase slippage, random genetic rearrangements and cloning difficulties. Here we report a newly designed synthetic TEF promoter to drive Cas9 expression, pTEF(-41–406)-Kozak, which is a rationally 3′-truncated version of the already known 5′-truncated pTEF(406) promoter of Y. lipolytica fused to a synthetic Kozak sequence. Our comparison of the promoters’ strength using hrGFP reporters and RT-qPCR showed that the synthetic pTEF(-41–406)-Kozak has an equivalent expression strength to that of pTEF(406), yet is at least 5 times stronger than the hybrid pUAS1B8-TEF(136). The pTEF(-41–406) promoter mediated high expression of Cas9 and was not associated with any growth defects. Moreover, expression of Cas9 under pTEF(-41–406)-Kozak increased the gene integration efficiency by up to 40 % relative to that when Cas9 is expressed under pUAS1B8-TEF(136). Both pTEF(-41–406) and pUAS1B8-TEF(136) performed equally well as drivers of Cas9 expression with respect to gene deletion as demonstrated both on the genotypic and phenoypic levels. This is the first study conducting rational 3′-truncation in TEF promoter in Y. lipolytica based on in silico analysis of promoter sequence and structure. This approach of promoter engineering can be extended to the engineering of other yeast promoters to generate small-sized synthetic biology parts for convenient engineering of biological systems. This work provides a strong Cas9 expression cassette for more convenient and efficient CRISPR-Cas9-mediated genome editing in Y. lipolytica which will facilitate harnessing the full potential of this industrial strain.