Gene Excision By Dual-Cutting CRISPR-Cas9 in Yarrowia Lipolytica

A genome editing tool has become increasingly required for metabolic engineering of non-model and non-conventional microorganism. For example, a CRISPR-Cas9 tool for the oleaginous yeast, Yarrowia lipolytica, has been used for promoting double-strand breaks. When provided with a homologous template, Cas9 activity at certain sites can lead to homologous recombination. In the absence of a homologous donor, indel mutations can occur leading to gene inactivation. However, as mutations are often the insertion or deletion of a single to three nucleotides, the activity of the edited gene may remain unaltered. Furthermore, the remaining unedited portions of targeted genes have proved problematic due to gene conversion events. Here, we describe the development and characterization of a gene excision tool for Yarrowia lipolytica. This tool has been demonstrated on five targets and has successfully removed segments as large as 4kb. The core principle underlying the gene excision methodology is the simultaneous double-stand break at two locations, and non-homologous end joining repair. Given that both breaks need to occur during the same cell division, and that these breaks have been repaired together, a 12-16% efficiency was greater than expected. Since the resulting genomic region has a significant size difference, screening by colony PCR is simple compared with alternative methods for identifying indels. Recent efforts to multiplex and improve the overall efficiency of this technology will be presented.