The present disclosure provides a fusion polypeptide comprising: a) an enzymatically active RNA-guided endonuclease that introduces a single-stranded break in a target DNA; and b) an error-prone DNA polymerase. The present disclosure provides a system comprising: a) a fusion polypeptide of the present disclosure; and b) a guide RNA. The present disclosure provides a cell comprising a fusion polypeptide of the present disclosure, or a system of the present disclosure. The present disclosure provides a method of mutagenizing a target polynucleotide.

The invention relates to a method for producing a ribonucleic acid (RNA) molecule composition comprising n different RNA molecule species, the method comprising a step of RNA in vitro transcription of a mixture of m different deoxyribonucleic acid (DNA) molecule species in a single reaction vessel in parallel, i.e. simultaneously, and a step of obtaining the RNA molecule composition. Also provided is the RNA composition provided by the inventive method and a pharmaceutical composition comprising the same as well as a pharmaceutical container. Moreover, the invention provides the RNA composition and the pharmaceutical composition for use as medicament.

This disclosure provides methods and landing pad constructs for generation of parental cell lines suitable for targeted integration. A method is provided by the parental cell line development; this is, the introduction of binding sites of BPV1 E2 protein to landing pad vectors so that expressed BPV1 E2 protein could locate the vector to transcriptionally active region in the genome. Cells with high expression level of reporter genes are selected for the next stage and will be used in the development of cell lines expressing another recombinant protein by recombination mediated cassette exchange (RMCE). Landing pad constructs include recombination target sites for site-specific recombinases, and therefore, it could be replaced with gene-of-interest expression construct containing the same set of recombination target sites. This yields the generation of producer cell lines with less effort compared to traditional cell line development by random integration.

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

The invention provides a single-stranded oligonucleotide represented by the formula [Xz — Lx].sub.m — X — Y — [Ly — Yz].sub.n, wherein X is represented by Xa-Xb, Xa is coupled with Y, and Xb and Y hybridize. Xa is composed of 1 to 40 nucleotides and contains at least one modified-nucleotide. Xb is composed of 4 to 40 nucleotides and contains at least one modified-nucleotide. Y is composed of 4 to 40 nucleotides and contains at least one ribonucleotide. Xz and Yz are composed of 5 to 40 nucleotides and contain at least one modified-nucleotide. Nucleotide sequences X, Xz and Yz have an antisense sequence capable of hybridizing with a target RNA. Lx and Ly are composed of 0 to 20 nucleotides.

Described herein are molecules for editing a cell. The molecules described herein generally comprise the following covalently-linked components and a nucleic acid encoding a guide RNA (gRNA) sequence targeting a target region in a cell and a region homologous to the target region comprising a change in sequence relative to the target region.

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

CRISPR/CAS-related compositions and methods for treatment of Sickle Cell Disease (SCD) are disclosed.

Provided are a composition for ribonucleoprotein delivery, comprising a guide RNA free of 5′-terminal phosphates, and a method for ribonucleoprotein delivery, using the same.

The invention relates to the field of molecular biology and cell biology. More specifically, the invention relates to CRISPR guide-RNA expression strategies for multiplex genome engineering.