Vinciane Borsenberger

CRISPR/Cas9 is a powerful tool to edit the genome of the yeast Yarrowia lipolytica. Here, we design a simple and robust method to knock-out multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs. We exemplify the potency of this approach by targeting the well-characterised acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family. We establish a correlation between the high lethality observed upon editing multiple loci and chromosomal translocations resulting from the simultaneous generation of several double-strand breaks (DSBs) and develop multiplex gene editing strategies. Using Homologous Directed Recombination to reduce chromosomal translocations, we demonstrated that simultaneous editing of four genes can be achieved and constructed a strain carrying a sextuple deletion of POX genes. We explore an "excision approach" by simultaneously performing two DSBs in genes, and reached 73 to 100% editing efficiency in double disruptions and 41.7 % in a triple disruption. This work led to identify SPS193 as a gene encoding a 2-4 dienoyl-CoA reductase, demonstrating the potential of this method to accelerate knowledge on gene function in expanded gene families.

The potentially prebiotic synthesis of ribo‐nucleotides by stepwise pyrimidine nucleobase assembly on arabinose‐3‐phosphate derivatives has been demonstrated in previous work [1]. Consideration of the provenance of pentose phosphates, by aldolisation or sugar phosphorylation, suggested that 2‐phosphate derivatives might be generated more easily than 3‐phosphate derivatives. In the 2‐phosphate series, nucleobase‐assembly chemistry to give ribo‐nucleotides/nucleic acid can be envisaged from xylo‐configured starting materials. In this paper, the derivation of xylose‐2‐phosphate derivatives by aldol chemistry and attempts to demonstrate subsequent pyrimidine nucleobase assembly are reported.