The invention provides compositions and methods for performing mammalian cell genetics, e.g., genetic screens, using near-haploid cells. The invention further provides genes and gene products isolated using the inventive methods and methods of use thereof.

Provided herein are CasX:gNA systems comprising CasX polypeptides, guide nucleic acids (gNA), and optionally donor template nucleic acids useful in the modification of a SOD1 gene. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the SOD1 protein or the SOD1 regulatory element. Also provided are methods of using such CasX:gNA systems to modify cells having such mutations and utility in methods of treatment of a subject with a SOD1-related disease.

The present disclosure provides bidirectional nucleic acid constructs that allow enhanced insertion and expression of a nucleic acid sequence of interest, e.g., encoding a therapeutic agent such as a polypeptide.

Disclosed herein are methods useful for modulating expression of a target gene by modulating binding between a ribonucleic acid (RNA) transcribed from at least one regulatory element of a target gene and a transcription factor which binds to both the RNA and the regulatory element. Also disclosed herein are methods and assays for identifying agents that interfere with binding between RNA transcribed from at least one regulatory element and a transcription factor which binds to the RNA and to the regulatory element.

Means and methods for preparing combinatorial libraries of DNA constructs, in particular expression cassettes, including nucleic acid constructs, expression vectors, host cells, methods for preparing host cells, and methods for producing polypeptides of interest, whereby the expression comprises an first intron and a second intron on either side of the polynucleotide to be expressed and a promoter and a terminator. Also claimed is a method of constructing eukaryotic host cells in which the cells are contacted with three polynucleotides and in which the first and second and the second and third are pairwise capable of homologous recombination and of subsequent formation of introns.

The invention features compositions and methods that are useful for identifying allele variants that modulate gene expression. Compositions and articles defined by the invention were isolated or otherwise manufactured.

The invention features compositions and methods that are useful for identifying allele variants that modulate gene expression. Compositions and articles defined by the invention were isolated or otherwise manufactured.

Disclosed herein are methods of identifying promoters that drive protein expression independently of methanol and are useful in driving protein expression in yeast. The method may comprise the steps of: fermenting yeast cells under at least one fermentation condition in the absence of methanol, collecting samples at different times during fermentation under the at least one fermentation conditions, determining the relative mRNA levels associated with native yeast genes in the samples, identifying one or more of the native yeast genes associated with higher than average levels of mRNA and determining putative promoters associated with the higher than average levels of mRNA encoding the native yeast genes, making expression constructs, each construct comprising one of the identified putative promoters and a gene encoding a marker protein and introducing the expression constructs into yeast cells, culturing the yeast cells comprising the expression constructs in the absence of methanol, determining marker protein expression by the cultured yeast cells and comparing marker protein expression driven by each of the putative promoters to identify promoters that drive protein expression independently of methanol and are useful in driving protein expression in yeast.

Aspects of the invention described herein relate to methods of making and using inducible promoters for transgene expression. The inducible promoters are derived from the NFAT-RE inducible system and are used to improve or enhance T cell survival and proliferation.

Embodiments disclosed herein provide a general, scalable, high-throughput, and high-resolution approach for experimental dissection of regulatory regions and driver nucleotides in the context of human biology and disease. Applicants present HiDRA, a novel high-resolution global screen for transcriptional regulatory activity in accessible chromatin regions, enabling high-efficiency, high-throughput, and high-resolution inference of regulatory activity.