Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.

Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.

Nanomechanical action opens endo-lysosomal compartments.

Endo-lysosomal escape is a highly inefficient process, which is a bottleneck for intracellular delivery of biologics, including proteins and nucleic acids. Herein, we demonstrate the design of a lipid-based nanoscale molecular machine, which achieves efficient cytosolic transport of biologics by destabilizing endo-lysosomal compartments through nanomechanical action upon light irradiation. We fabricate lipid-based nanoscale molecular machines, which are designed to perform mechanical movement by consuming photons, by co-assembling azobenzene lipidoids with helper lipids. We show that lipid-based nanoscale molecular machines adhere onto the endo-lysosomal membrane after entering cells. We demonstrate that continuous rotation-inversion movement of Azo lipidoids triggered by ultraviolet/visible irradiation results in the destabilization of the membranes, thereby transporting cargoes, such as mRNAs and Cre proteins, to the cytoplasm. We find that the efficiency of cytosolic transport is improved about 2.1-fold, compared to conventional intracellular delivery systems. Finally, we show that lipid-based nanoscale molecular machines are competent for cytosolic transport of tumour antigens into dendritic cells, which induce robust antitumour activity in a melanoma mouse model.

Lipidoid Artificial Compartments for Bidirectional Regulation of Enzyme Activity through Nanomechanical Action.

Photoresponsive inhibitor and noninhibitor systems have been developed to achieve on-demand enzyme activity control. However, inhibitors are only effective for a specific and narrow range of enzymes. Noninhibitor systems usually require mutation and modification of the enzymes, leading to irreversible loss of enzymatic activities. Inspired by biological membranes, we herein report a lipidoid-based artificial compartment composed of azobenzene (Azo) lipidoids and helper lipids, which can bidirectionally regulate the activity of the encapsulated enzymes by light. In this system, the reversible photoisomerization of Azo lipidoids triggered by UV/vis light creates a continuous rotation-inversion movement, thereby enhancing the permeability of the compartment membrane and allowing substrates to pass through. Moreover, the membrane can revert to its impermeable state when light is removed. Thus, enzyme activity can be switched on and off when encapsulating enzymes in the compartments. Importantly, since neither mutation nor modification is required, negligible loss of activity is observed for the encapsulated enzymes after repeated activation and inhibition. Furthermore, this approach provides a generic strategy for controlling multiple enzymes by forgoing the use of inhibitors and may broaden the applications of enzymes in biological mechanism research and precision medicine.

Effects of combined enzymatic hydrolysis and fed-batch operation on efficient improvement of ferulic acid and p-coumaric acid production from pretreated corn straws.

In the present work, the effects of combined enzymatic hydrolysis by cellulase and xylanase (CXEH), fed-batch enzymatic hydrolysis (FBEH) operation and kinetics on production of ferulic acid (FA) and p-coumaric acid (pCA) from pretreated corn straws were investigated. The results showed that CXEH could efficiently increase production of FA and pCA. When performed the FBEH operation by feeding 150 mL enzymatic hydrolysis solution (1.5 % enzyme concentration, 5:4 (v/v) ratio of cellulase to xylanase and 2.0 % substrate loading) to 250 mL batch enzymatic hydrolysis solution at 36 h, the maximum production (2178.58 and 2710.17 mg/L) and production rate (590.95 and 727.89 mg/L.h) of FA and pCA were respectively obtained. Moreover, the disruption of fiber tissues, enhancement of crystallinity and accelerated degradation of hemicelluloses and lignocelluloses caused by CXEH contributed to effectively improving production of FA and pCA in corn straws.

Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial Clostridium community.

Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single-specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5-5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169-(pXY1-Pthl -augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5-fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.

Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous ß-carotene production.

ß-Carotene is a terpenoid molecule with high hydrophobicity that is often used as an additive in foods and feed. Previous work has demonstrated the heterologous biosynthesis of ß-carotene from an intrinsic high flux of acetyl-CoA in 12 steps through 11 genes in Yarrowia lipolytica. Here, an efficient biosynthetic pathway capable of producing 100-fold more ß-carotene than the baseline construct was generated using strong promoters and multiple gene copies for each of the 12 steps. Using fed-batch fermentation with an optimized medium, the engineered pathway could produce 4g/L ß-carotene, which was stored in lipid droplets within engineered Y. lipolytica cells. Expansion of these cells for squalene production also demonstrated that Y. lipolytica could be an industrially relevant platform for hydrophobic terpenoid production.

Production of ß-carotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica.

OBJECTIVES: To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene ß-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce ß-carotene. RESULTS: To increase the integration efficiency of a 3.8 kb carS under the control of P GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg ß-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica. CONCLUSION: Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to ß-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.

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

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.

One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica.

Yarrowia lipolytica is an unconventional yeast, and is generally recognized as safe (GRAS). It provides a versatile fermentation platform that is used commercially to produce many added-value products. Here we report a multiple fragment assembly method that allows one-step integration of an entire ß-carotene biosynthesis pathway (~11 kb, consisting of four genes) via in vivo homologous recombination into the rDNA locus of the Y. lipolytica chromosome. The highest efficiency was 21%, and the highest production of ß-carotene was 2.2 ± 0.3 mg per g dry cell weight. The total procedure was completed in less than one week, as compared to a previously reported sequential gene integration method that required n weeks for n genes. This time-saving method will facilitate synthetic biology, metabolic engineering and functional genomics studies of Y. lipolytica.