The disclosure provides novel methods and compositions for gene editing. In particular, the disclosure relates to compositions and methods of making nucleic acid donor templates for highly efficient and precise gene editing.

The invention belongs to the technical field of genetic engineering, and particularly to a type I-F CRISPR-Cas system based on Zymomonas mobilis (Z. mobilis) including: four CRISPR structural sequences and one cas gene cluster, wherein the cas gene cluster comprises casi gene, cas3 gene, csyl gene, csy2 gene, csy3 gene and csy4 gene, wherein the cast-3 gene is a fusion form fused by a cast gene and a cas3 gene. The purpose of the present invention is to use Z. mobilis as a model bacterium, using a CRISPR-Cas system encoded by the genome of the Z. mobilis and exogenous CRISPR-Cas12a system to build a genome editing platform so as to provide a set of powerful tools for carrying out basic and applied research in this bacterium and similar cells, and promoting the development of metabolic engineering, systems biology and synthetic biology.

The present disclosure provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides novel class 1, Type IV and novel class I, Type I Cas proteins and their use in modifying target sequences.

Compositions and methods concern the sequence modification of an endogenous genomic DNA region. Certain aspects relate to a method for site-specific sequence modification of a target genomic DNA region in cells comprising: contacting the cells with an activating composition; transfecting the cells with a transfection composition comprising (a) donor DNA and (b) a DNA digesting agent; wherein the donor DNA comprises: (i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region; and (ii) a sequence modification region; and wherein the genomic DNA sequence is modified specifically at the target genomic DNA region.

The present invention relates to a method for producing a panel of cells (i.e. a cell library) expressing various different mutant variants of a protein of interest, wherein only one of said mutant variants is expressed per cell from a single gene copy. The present invention also relates to a method or cell library for identifying a mutant variant of a protein of interest having a different or modified biological activity as compared to the corresponding wild-type protein of interest. According to the present invention the identified mutant variant of a protein of interest may be applied for white biotechnology.

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 invention relates to variant AAV capsid polypeptides, wherein the variant AAV capsid polypeptides exhibit increased transduction and/or tropism in human pancreatic tissue or human islets as compared non-variant parent capsid polypeptides.

The present disclosure relates to a method of editing a genome based on the CRISPR/Cpf1 system and a use thereof, and the CRISPR system using an oligonucleotide-induced mutation and 3′-truncated crRNA according to the present disclosure provides the significant effect of genome editing to the target DNA and thus it is expected that the CRISPR system of the present disclosure may be used in a wide range of fields such as a composition for gene editing using gene scissors, screening at the genome level, therapeutics for various diseases including cancer, development of a composition for disease diagnosis or imaging, and development of transgenic animals and plants.

The present disclosure provides compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The nucleotide change can include a single-nucleotide change (e.g., any transition or any transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap, which is homologous to a strand of the targeted endogenous DNA sequence to be edited, but which contains the desired one or more nucleotide changes and which, following synthesis by the polymerase (e.g., reverse transcriptase), becomes incorporated into the target DNA molecule. Also disclosed herein are various methods that leverage prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion disease through the installation of protection mutations, manipulating RNA-encoding genes for the installation of RNA tags for controlling the function and expression of RNA, using prime editing to construct sophisticated gene libraries, using prime editing to insert immunoepitopes into proteins, use of prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.

Related are a recombinant microorganism for producing L-valine, a construction method and an application thereof. Through transferring an amino acid dehydrogenase gene and/or activating activity of a transhydrogenase and/or a NAD kinase, reducing power of NADPH in cell is increased, the titer and yield of L-valine generated by Escherichia coli are improved, and the production of L-valine by one-step anaerobic fermentation is achieved.