Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for engineering Cas9 and Cas9 variants that have increased activity on target sequences that do not contain the canonical PAM sequence. In some embodiments, fusion proteins comprising such Cas9 variants and nucleic acid editing domains, e.g., deaminase domains, are also provided.

Provided herein are compositions and methods for improving the genome-wide specificities of targeted base editing technologies.

Embodiments provide a modified microbe capable of growing in or fermenting a solution, or lignocellulosic hydrolysate, comprising ferulic acid and/or coniferyl aldehyde. The microbe has one or more modifications to provide: (a) a decrease in copy number or expression of a BNA7 gene; (b) an increase in copy number or expression of one or more pentose phosphate pathway genes; and/or (c) localization of one or more products of the pentose phosphate pathway genes to the mitochondria or endoplasmic reticulum. Also provided is a microbe having modified expression or copy number of BNA7 and/or one or more of the pentose phosphate pathway genes. The pentose phosphate pathway genes may in certain embodiments be selected from at least one of ZWF1, TKL1, RPE1 and GND1. Also provided is a method for fermenting a substrate comprising ferulic acid and/or coniferyl aldehyde to produce a fermentation product.

A gene ansB knockout mutant of Citrobacter werkmanii and an application thereof are provided. The gene ansB knockout mutant of the C. werkmanii is C. werkmanii with a gene ansB knocked out and a nucleotide sequence of the gene ansB is as shown in SEQ ID NO: 1. In the present invention, the acquired engineering bacteria with the gene ansB of the C. werkmanii knocked out are cultured in LB, TSB, NB and other media at 25° C. and 30° C., so that a biofilm formation capacity of the C. werkmanii on a polypropylene material is improved. Thus, the application scenarios and scopes of the C. werkmanii in heavy metal ion adsorption and construction of cellular protein synthesis micro-factories are broadened.

A gene ansB knockout mutant of Citrobacter werkmanii and an application thereof are provided. The gene ansB knockout mutant of the C. werkmanii is C. werkmanii with a gene ansB knocked out and a nucleotide sequence of the gene ansB is as shown in SEQ ID NO: 1. In the present invention, the acquired engineering bacteria with the gene ansB of the C. werkmanii knocked out are cultured in LB, TSB, NB and other media at 25° C. and 30° C., so that a biofilm formation capacity of the C. werkmanii on a polypropylene material is improved. Thus, the application scenarios and scopes of the C. werkmanii in heavy metal ion adsorption and construction of cellular protein synthesis micro-factories are broadened.

The present disclosure is directed to methods of inducing rejuvenation in a population of adult glial progenitor cells, and methods of treating a subject having a myelin deficiency. The method of inducing rejuvenation in a population of adult glial progenitor cells, may comprise: administering, to the population of adult glial progenitor cells, one or more nucleic acid molecules encoding microRNAs, wherein administering suppresses the signal transducer and activator of transcription 3 (STAT3) signaling pathway; and/or administering microRNAs, wherein administering suppresses the E2F transcription factor 6 (E2F6) signaling pathway; and/or administering microRNAs, wherein administering suppresses the Myc-associated factor X (MAX) signaling pathway, wherein said one or more nucleic acid molecules are administered in an amount sufficient to induce rejuvenation in the population of adult glial progenitor cells.

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered microorganisms. The present invention describes engineered microorganisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These microorganisms can dephosphorylate N-acetylglucosamine-6-phosphate to N-acetylglucosamine and convert the N-acetylglucosamine to N-acetylmannosamine. These microorganisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the present invention provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose, N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phosphate to N-acetylglucosamine, converts N-acetylglucosamine to N-acetylmannosamine and convert the latter further to N-acetyl-neuraminate.

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered microorganisms. The present invention describes engineered microorganisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These microorganisms can dephosphorylate N-acetylglucosamine-6-phosphate to N-acetylglucosamine and convert the N-acetylglucosamine to N-acetylmannosamine. These microorganisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the present invention provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose, N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phosphate to N-acetylglucosamine, converts N-acetylglucosamine to N-acetylmannosamine and convert the latter further to N-acetyl-neuraminate.

Embodiments disclosed include systems, devices, and methods for analysis of samples containing particles used for gene delivery to determine a quality of the sample and/or an indication that the gene delivery particles are in a full, partial, and/or empty state. The present disclosure also relates to determining a protein and/or NA content in samples with known proportions of gene delivery particles in a full, partial, and/or empty state and based on the determination, establish a relationship between NA content and proportions of gene delivery particles in a full state. The present disclosure also relates to using such an established relationship to predict a proportion of the gene delivery particles in a full, partial, and/or empty state in test samples having the gene delivery particles in an unknown state.

The present invention discloses rapid and cost-effective method of de-glycosyation of a glycoprotein, wherein, glycoprotein is combined with anionic surfactant and reducing agent and non-ionic surfactant in order to obtain stable denatured glycoprotein. An endoglycosidase is further added to denatured glycoprotein to cleave N-linked glycans in order to obtain de-glycosylated protein. A rapid tool for assessing the protein conformation by partial de-glycosylation is also presented wherein the partial de-glycosylated protein is analysed using capillary electrophoresis (CE-SDS).