Gene Excision by Dual-Guide CRISPR-Cas9.

CRISPR-Cas9 is frequently used for creating double-strand DNA breaks that result in indels through non-homologous end joining. Indels can revert to wild-type sequence and require sequencing or complex assays to measure. Cutting by two guide RNAs can lead to single indels at either cut site or simultaneous cutting at both sites and repair leading to gene excision.

Simultaneous Gene Excision and Integration by Dual-Guide CRISPR-Cas9.

Metabolic engineering frequently requires both gene knockouts and gene integration. CRISPR-Cas9 has been extensively used to create double-stranded DNA breaks that result in indel mutations; however, such mutations can revert or create toxic product. Gene integration can also be accomplished by CRISPR-Cas9 introduced double-stranded DNA breaks and a donor DNA cassette.

Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica.

Yarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR-Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y.

Oleaginous yeast for biofuel and oleochemical production.

Current transportation fuels derived from petroleum can also be made from microbial systems. In particular, oleaginous yeast have naturally evolved high flux pathways for fatty acids in the form of neutral lipids, which can be converted into a variety of drop-in fuels. Here, we describe the recent advances in the use of the four most popular oleaginous yeasts for making lipids and other potential fuels - Yarrowia lipolytica, Lipomyces starkeyi, Rhodosporidium toruloides, and Cutaneotrichosporon oleaginosus.

Alternative Substrate Metabolism in .

Recent advances in genetic engineering capabilities have enabled the development of oleochemical producing strains of . Much of the metabolic engineering effort has focused on pathway engineering of the product using glucose as the feedstock; however, alternative substrates, including various other hexose and pentose sugars, glycerol, lipids, acetate, and less-refined carbon feedstocks, have not received the same attention. In this review, we discuss recent work leading to better utilization of alternative substrates.

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest.

Engineering yeast for utilization of alternative feedstocks.

Realizing the economic benefits of alternative substrates for commodity chemical bioproduction typically requires significant metabolic engineering of common model organisms, such as Saccharomyces cerevisiae. A growing toolkit is enabling engineering of non-conventional yeast that have robust native metabolism for xylose, acetate, aromatics, and waste lipids. Scheffersomyces stipitis was engineered to produce itaconic acid from xylose.