L+SScF 232 ABSTRACT 1 A techno-economic analysis was conducted for a simplified lignocellulosic ethanol 2 production process developed and proven by the University of Florida at laboratory, pilot, and 3 demonstration scales. Data obtained from all three scales of development were used with 4 Aspen Plus to create models for an experimentally-proven base-case and 5 hypothetical 5 scenarios. The model input parameters that differed among the hypothetical scenarios were 6 time of L+SScF, enzyme loading, enzymatic conversion, solids loading, and overall process 7 yield. The minimum ethanol selling price (MESP) varied between 50.38 and 62.72 US 8 cents/L. The feedstock and the capital cost were the main contributors to the production cost, 9 comprising between 23-28% and 40-49% of the MESP, respectively. A sensitivity analysis 10 showed that overall ethanol yield had the greatest effect on the MESP. These findings suggest 11 that future efforts to increase the economic feasibility of a cellulosic ethanol process should 12 focus on optimization for highest ethanol yield.

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein–tagged genes.

AbstractHere we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes