The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods.

Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, visualization of a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs, trans-activating CRISPR RNAs, guide RNAs, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided are RGN systems for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.

The invention relates to methods, kits, and compositions for reducing the level of or eliminating a nucleic acid vector in situ. The invention encompasses compositions and methods for selectively eradicating nucleic acid vectors in the microbiota using packaged phagemids. The microbiota can be intestinal and the packaged phagemids can be administered orally. The phagemid encodes a nuclease or other enzyme that genetically modifies the nucleic acid vector so that the nucleic acid vector can be inactivated or eliminated.

Compositions and methods for targeting pre-determined DNA sequences in bacterial cells are provided. The methods result in the targeted elimination of bacterial cells that comprise the pre-determined DNA sequence(s). Compositions comprise DNA constructs comprising nucleotide sequences that encode a Cms1 protein operably linked to a promoter that is operable in the cells of interest. Methods to use these DNA constructs to selectively target and eliminate bacterial cells that harbor the targeted DNA sequence(s) are described herein.

The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to a gene editing system derived from Flavobacterium and uses thereof.

Methods for modification of target nucleic acids. The method involves a construct in which guide RNA is covalently linked to donor RNA (fusion NA) to be introduced into the target nucleic acid by homologous recombination and is based on the introduction of a nuclease, e.g. CRISPR or TALEN, into the cell containing the target nucleic acid. The fusion NA may be introduced as a DNA vector.

Described herein are materials and methods for user-controlled adjustment of plasmid copy numbers.

Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.

An expression system and process for the production of Diphtheria toxin polypeptides or mutated forms thereof, such as the toxoid CRM197 polypeptide, in genetically-modified E. coli with high yield is described. The system and process is based on the uncoupling of biomass growth from recombinant protein induction, i.e. using an inducer of protein production that cannot be used as a carbon source for growth by the bacteria. The use of specific components and conditions that improve protein yields are also described.

Described herein are methods and vectors for rational, multiplexed manipulation of chromosomes within open reading frames (e.g., in protein libraries) or any segment of a chromosome in a cell or population of cells, in which various CRISPR systems are used.