Disclosed are transformed cells and related nucleotide and protein sequences, and fermentation compositions and methods, all of which are related to providing selective advantage in fermentation. For example, a selective advantage results from transformation of a cell with a nucleic acid that allows a transformed cell to metabolize one or more nitrogen-, phosphorous-, and/or sulfur-containing compounds that a native cell of the same species as the transformed cell cannot metabolize, and from fermentation of the transformed cell using one or more feedstocks, such as fractioned grain, which are depleted in or free of conventional nitrogen-, phosphorous-, and/or sulfur-containing compounds that a native cell of the same species as the transformed cell can metabolize. Also disclosed are methods for improved oxygen transfer in an aerobic or microaerobic fermentation.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

Methods for reversing one or more mutations in the telomerase (TERT) promoter are provided and may be used to treat a cancer. In some embodiments, programmable base editing (PBE) is used to correct a mutated TERT promoter (e.g., −124 C>T, −228C>T, or −250C>T to −124 C, −228C, or −250C, respectively) by using a single guide (sg) RNA-guided and deactivated Campylobacter jejuni Cas9-fused adenine base editor (CjABE). These methods can be used to treat a cancer, such as for example glioblastoma multiforme (GBM), in a mammalian subject in vivo.

A protein antigen combination for detecting Alzheimer's disease autoantibodies in human serum sample and an application thereof are disclosed. The protein antigen combination includes at least two protein fragments selected from the following proteins: MAPT, ADARB1, P21, DNAJC8, RAGE, ASXL1, and JMJD2D. Based on the protein antigen composition, a kit for diagnosis, especially for early diagnosis of Alzheimer's disease or its related risks, can be prepared. By using the protein antigen composition provided by the invention, early Alzheimer's disease can be quickly and accurately diagnosed, which has important practical significance.

Disclosed are RNA site-directed editing using artificially constructed RNA editing enzymes and related uses. Provided is the fusion of an RNA recognition domain for binding RNA and a functional effector domain to form a new functional protein. The new functional protein specifically targets target RNA by means of the recognition domain and performs RNA editing using the effector domain.

Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

The present disclosure provides highly multiplexed base editing methods and compositions that minimize the induction of DNA damage sensors in eukaryotic cells and maintain cell viability. The disclosed base editing methods improve the survival of eukaryotic cells after large-scale genome editing. These methods are based upon the discovery that use of a dead Cas9 base editor and optimal cell conditions during and after base editing enhances cells' tolerance to and survival following thousands of edits to the genome. Optimal cell conditions after base editing include the use of a combination of small molecule factors and/or inhibitors. These methods are facilitated by the design and use of tens to hundreds to thousands of gRNAs for guiding the base editor to the target sequences. The disclosed methods are capable of inducing between ten and 300,000 edits to the genome of a eukaryotic cell. Further disclosed are pharmaceutical compositions and compositions of eukaryotic cells comprising fusion proteins and a plurality of unique gRNAs, and a combination of small molecule factors and inhibitors. Also disclosed are kits for the generation of the fusion protein-gRNA complexes described herein.

The present invention relates to a fusion protein for enhancing gene editing and use thereof. In particular, the invention provides an enhanced fusion protein. The enhanced fusion proteins of the present invention can significantly increase gene editing efficiency in vivo or in vitro as compared to the wildtype gene editing protein.

The present disclosure provides methods of introducing site-specific mutations in a target cell and methods of determining efficacy of enzymes capable of introducing site-specific mutations. The present disclosure also provides methods of providing a bi-allelic sequence integration, methods of integrating of a sequence of interest into a locus in a genome of a cell, and methods of introducing a stable episomal vector in a cell. The present disclosure further provides methods of generating a human cell that is resistant to diphtheria toxin.

The present invention relates to a base editing compound comprising or consisting of (a) a Cas protein, and, covalently connected therewith; (b) a nucleobase-modifying enzyme, wherein the covalent connection of (a) and (b) is (i) direct; (ii) provided by a peptide comprising at least one Pro residue, said peptide having a length between 1 and 20 preferably between 1 and 15 amino acids; or (iii) provided by a non-peptidic linker, said non-peptidic linker being a small organic molecule comprising one or more double bonds, one or more triple bonds, and/or one or more aromatic rings.