Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

Abstract

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.

CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica

“…Over the past 5 years, the type II CRISPR‐Cas9 system from Streptococcus pyogenes has been adapted for genome editing and transcriptional regulation in a wide range of organisms (DiCarlo et al, ; Gao et al, ; Jacobs, Ciccaglione, Tournier, & Zaratiegui, ; Jinek et al, ; Mali et al, ; Pohl, Kiel, Driessen, Bovenberg, & Nygard, ). Targeted DSB from Cas9 endonuclease complexed with a targeting single guide RNA (sgRNA) are repaired by NHEJ, often causing an indel mutation and disrupting gene function.…”

History of genome editing in yeast

“…Another group (Gao et al, ) created a Y. lipolytica codon‐optimized Cas9 and used a single plasmid system to deliver the gRNA in order to test the efficiency of NHEJ and HR. After 4 days of outgrowth, 86, 37 and 19% efficiency was achieved for one, two or three targeted genes, respectively, when no donor DNA was present.…”

Recent Advances in Metabolic Engineering of Yarrowia lipolytica for Lipid Overproduction

“…[29] In order to increase the HR rate, the KU70 gene, which is involved in the NHEJ pathway, was deleted using the Cre-lox system, which increased the HR rate from less than 0.5% to 33-71%. However, in contrast to Saccharomyces cerevisiae, the nonhomologous endjoining (NHEJ) recombination pathway and not the HR pathway is mainly responsible for the repair of double-stranded DNA breaks in Y. lipolytica.…”

“…[45] Generally, expression/deletion cassettes bounded by lox sequences were constructed to perform marker rescue the functional analysis of gene families in Y. lipolytica via the expression of Cre recombinase. [29] CRISPR-Cas9 technology also greatly increased the HR rate for genome integration, which reached up to 64% in WT Y. lipolytica and up to 100% in a ΔKU70 strain. The CRISPR-Cas9 system can induce targeted DNA doublestranded breaks, which in turn can be repaired either via HR in the presence of homologous donor DNA, or via NHEJ without donor DNA.…”

“…[3] In Y. lipolytica, the de novo lipid synthesis pathway is induced by excess carbon and exhaustion of other essential nutrients from the culture medium. [29] In this review, we focus on the lipid metabolism of Y. lipolytica, the available genetic tools, and the latest progress in manipulating this organism for the overproduction of various lipid-derived chemicals. [6,19] The neutral lipid mostly consist of triacylglyceride (TAG) (about 95%) and sterol esters (less than 5%), which are not suitable for incorporation into the membrane phospholipid bilayer, and therefore cluster in the cytoplasm to form so-called lipid bodies.…”