Multiple Parameters Drive the Efficiency of CRISPR/Cas9-Induced Gene Modifications in Yarrowia lipolytica

Yarrowia lipolytica is an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR-Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5' end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation in Y. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editing Y. lipolytica genome. Finally, the optimized system was used to promote in vivo directed mutagenesis via homology-directed repair with a ssDNA oligonucleotide.