Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.

Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition.

Non-productive binding of cellulases to lignin inhibits enzymatic hydrolysis of biomass, increasing enzyme requirements and the cost of biofuels. This study used site-directed mutagenesis of the Trichoderma Cel7A carbohydrate binding module (CBM) and linker to investigate the mechanisms of adsorption to lignin and engineer a cellulase with increased binding specificity for cellulose. CBM mutations that added hydrophobic or positively charged residues decreased the specificity for cellulose, while mutations that added negatively charged residues increased the specificity. Linker mutations that altered predicted glycosylation patterns selectively impacted lignin affinity. Beneficial mutations were combined to generate a mutant with 2.5-fold less lignin affinity while fully retaining cellulose affinity. This mutant was uninhibited by added lignin during hydrolysis of Avicel and generated 40% more glucose than the wild-type enzyme from dilute acid-pretreated Miscanthus. Biotechnol. Bioeng. 2016;113: 1369-1374. © 2015 Wiley Periodicals, Inc.

Structural insights into the affinity of Cel7A carbohydrate-binding module for lignin.

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.

Evaluating endoglucanase Cel7B-lignin interaction mechanisms and kinetics using quartz crystal microgravimetry.

The kinetics and mechanisms of protein interactions with solid surfaces are important to fields as diverse as industrial biocatalysis, biomedical engineering, food science, and cell biology. The nonproductive adsorption of cellulase enzymes to lignin, a plant cell wall polymer, reduces their effectiveness in saccharifying biomass. Cellulase has been shown to interact with lignin, but the heterogeneity of lignin surfaces, challenges in measuring irreversible components of these interactions, and fast adsorption rates make quantifying the reaction kinetics difficult. This work employs quartz crystal microgravimetry with dissipation monitoring (QCM-D) for real-time measurement of adsorbed mass on a flat lignin surface. We have developed a method for casting homogeneous lignin films that are chemically similar to lignin found in pretreated biomass, and used QCM-D to compare three models of reversible-irreversible binding behavior: a single-site transition model, a transition model with changing adsorbate footprint, and a two-site transition model. Of the three models tested, the two-site transition model provides the only kinetic mechanism able to describe the behavior of Cel7B binding to lignin. While the direct implications of lignin-cellulase interactions may be limited to biomass deconstruction for renewable energy and green chemistry, the analytical and experimental methods demonstrated in this work are relevant to any system in which the kinetics and reaction mechanism of reversible and irreversible protein adsorption at a solid-liquid interface are important.

The role of microsphere fabrication methods on the stability and release kinetics of ovalbumin encapsulated in polyanhydride microspheres.

The effect of microsphere fabrication methods on the stability and release kinetics of ovalbumin encapsulated in polyanhydride microspheres was investigated. The polyanhydrides used were poly(sebacic anhydride) (poly(SA)) and a 20:80 random copolymer of poly[1,6-bis(p-carboxyphenoxy)hexane] (poly(CPH)) and poly(SA). Microspheres were fabricated using three double emulsion methods (water/oil/water, water/oil/oil and solid/oil/oil) and cryogenic atomization. The encapsulation efficiency was highest for cryogenic atomization and lowest when the w/o/w technique was used. Microspheres fabricated by the s/o/o method had the largest initial burst of released protein. All the methods resulted in zero-order release of the protein after the burst. The release of ovalbumin from poly(SA) and 20:80 (CPH:SA) microspheres lasted approximately 3 and approximately 6 weeks, respectively. For all fabrication methods the primary structure of released ovalbumin was conserved as determined by gel electrophoresis. The secondary structure of ovalbumin encapsulated in 20:80 (CPH:SA) w/o/w microspheres was not conserved.