Compact CRISPR genetic screens enabled by improved guide RNA library cloning.

CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Ionic Liquid Tolerance of Yeasts in Family Dipodascaceae and Genus Wickerhamomyces.

In previous studies of ionic liquid (IL) tolerance of numerous species of ascomycetous yeasts, two strains of Wickerhamomyces ciferrii and Galactomyces candidus had unusually high tolerance in media containing up to 5% (w/v) of the 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]). The study aimed at investigating whether additional strains of these species, and additional species in the Dipodascaceae family, also possess IL tolerance, and to compare sensitivity to the acetate and chloride versions of the ionic liquid. Fifty five yeast strains in the family Dipodascaceae, which encompasses genera Galactomyces, Geotrichum, and Dipodascus, and seven yeast strains of species Wickerhamomyces ciferrii were tested for ability to grow in laboratory medium containing no IL, 242 mM [C2C1Im][OAc], or 242 mM [C2C1Im]Cl, and in IL-pretreated switchgrass hydrolysate. Many yeasts exhibited tolerance of one or both ILs, with higher tolerance of the chloride anion than of the acetate anion. Different strains of the same species exhibited varying degrees of IL tolerance. Galactomyces candidus, UCDFSTs 52-260, and 50-64, had exceptionally robust growth in [C2C1Im][OAc], and also grew well in the switchgrass hydrolysate. Identification of IL tolerant and IL resistant yeast strains will facilitate studies of the mechanism of IL tolerance, which could include superior efflux, metabolism or exclusion.

Conversion of cassava leaf to bioavailable, high-protein yeast cell biomass.

BACKGROUND: Cassava leaves are an abundant global agricultural residue because the roots are a major source of dietary carbohydrates. Although cassava leaves are high in protein, the protein is not bioavailable. This work aimed to convert cassava leaves to a bioavailable protein-rich animal feed ingredient using high-protein yeasts. RESULTS: The structural proteins (ca 200 g kg-1 d.b.) from sundried cassava leaves were solubilized by mild alkali pretreatment, and the resulting cassava leaf hydrolysate (CLH) was used to screen for growth of 46 high-protein yeasts from 30 species. Promising candidates from the initial screen cultivated at a 10 mL scale demonstrated increases in relative abundance of essential amino acids over that of CLH. In particular, lysine, growth-limiting for some livestock, was increased up to 226% over the CLH content. One yeast, Pichia kudriavzevii UCDFST 11-602, was grown in 3 L of CLH in a bioreactor to examine the scale-up potential of the yeast protein production. While glucose was completely consumed, yeast growth exited log phase before depleting either carbon or nitrogen, suggesting other growth-limiting factors at the larger scale. CONCLUSIONS: High-value animal feed with enriched essential amino acid profiles can be produced by yeasts grown on agricultural residues. Yeasts convert structural protein solubilized from cassava leaves to essential amino acid-enriched, digestible protein. The low carbohydrate content of the leaves (ca 200 g kg-1 d.b.), however, necessitated glucose supplementation for yeast growth. © 2018 Society of Chemical Industry.

1-Ethyl-3-methylimidazolium tolerance and intracellular lipid accumulation of 38 oleaginous yeast species.

Pretreatment with ionic liquids (IL) such as 1-ethyl-3-methylimidazolium chloride or acetate is an effective method for aiding deconstruction of lignocellulosic biomass; however, the residual IL remaining in hydrolysates can be inhibitory to growth of ethanologenic or oleaginous yeasts that have been examined in the literature. The aim of this study was to identify oleaginous yeasts that are tolerant of the IL [C2C1Im][OAc] and [C2C1Im]Cl using 45 strains belonging to 38 taxonomically diverse species within phyla Ascomycota and Basidiomycota. Yeasts were cultivated in laboratory medium supplemented with 0, 2, or 4% IL in 96-well plates. The eight most tolerant strains were then cultivated in 10-mL media with no IL, 242mM [C2C1Im][OAc], or 242mM [C2C1Im]Cl. The effects of [C2C1Im]+ exposure on cell mass production and lipid accumulation varied at the species and strain level. The acetate salt decreased cell biomass and lipid production more severely than did the chloride ion for six strains. Lipid output was not markedly different (2.1 vs. 2.3 g/L) in Yarrowia lipolytica UCDFST 51-30, but decreased from 5 to 65% in other yeasts. An equimolar concentration of the chloride salt resulted in much milder effects, from 25% decrease to 66% increase in lipid output. The highest lipid outputs in this media were 8.3 and 7.9 g/L produced by Vanrija humicola UCDFST 10-1004 and UCDFST 12-717, respectively. These results demonstrated substantial lipid production in the presence of [C2C1Im]Cl at concentrations found in lignocellulosic hydrolysates, and thus, these two strains are ideal candidates for further investigation.