A highly efficient ethanol-fermentative yeast having high efficiency in ethanol production is provided without introducing a foreign gene. The highly efficient ethanol-fermentative yeast features a fermentative yeast effectively producing ethanol from pentose and hexose and being deposited to NITE Patent Microorganisms Depositary under the accession number NITE BP-01963.

The present invention provides high efficiency targeted and marker-less single or simultaneous multiple integrations using nucleases and a stable plasmid in Kluyveromyces host cells.

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.

The present disclosure provides compositions and methods to increase the percentage of edited cells in a cell population when employing nucleic-acid guided editing, as well as automated multi-module instruments for performing these methods. Specifically, the disclosure relates to methods, compositions, modules and automated multi-module cell processing instruments that increase the efficiency of nucleic acid-guided editing in a cell population using a nucleic acid nuclease (i.e., an RNA-guided nuclease or “RGN”)/single-strand binding protein (“SSB”) fusion system. The system leverages a single-strand binding protein (SSP) and single-strand DNA annealing protein (“SSAP”) interactions to drive enhanced recruitment.

Rationally-designed LAGLIDADG meganucleases and methods of making such meganucleases are provided. In addition, methods are provided for using the meganucleases to generate recombinant cells and organisms having a desired DNA sequence inserted into a limited number of loci within the genome, as well as methods of gene therapy, for treatment of pathogenic infections, and for in vitro applications in diagnostics and research.

The present invention provides for a genetically modified yeast host cell capable of producing elevated levels of 3-methyl-3-butene-1-ol or isoprenol.

The present invention relates to polypeptides which are Gal2 variants comprising at least one amino acid substitution at a position corresponding to T354, and optionally further amino acid substitution(s). The present invention further relates to nucleic acid molecules encoding the polypeptides and to host cells containing said nucleic acid molecules. The present invention further relates to a method for the production of bioethanol and/or other bio-based compounds, comprising the expression of said nucleic acid molecules, preferably in said host cells. The present invention also relates to the use of the polypeptides, nucleic acids molecule or host cells for the production of bioethanol and/or other bio-based compounds, and/or for the recombinant fermentation of biomaterial containing pentose(s), preferably D-xylose and/or L-arabinose.

Methods and compositions related to a nucleic acid-guided nuclease cell targeting screen are provided. The invention relates to compositions and methods for identifying cell targeting proteins that, when associated with a nucleic acid-guided nuclease (such as Cas9), enables at least the nucleic acid-guided nuclease to be targeted to the surface of a target cell or internalized by a target cell, i.e., a cell targeted by the cell targeting agent.

Non-conventional yeasts are disclosed herein comprising at least one RNA-guided endonuclease (RGEN) comprising at least one RNA component that does not have a 5′-cap. This uncapped RNA component comprises a sequence complementary to a target site sequence in a chromosome or episome in the yeast. The RGEN can bind to, and optionally cleave, one or both DNA strands at the target site sequence. An example of an RGEN herein is a complex of a Cas9 protein with a guide RNA. A ribozyme is used in certain embodiments to provide an RNA component lacking a 5′-cap. Further disclosed are methods of genetic targeting in non-conventional yeast.

The present invention relates to a fusion protein comprising a Cas9 domain and a Spo11 domain, as well as the use of this protein to induce targeted meiotic recombinations in a eukaryotic cell.