Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.

Described herein are method for generating a vector for editing a cell. The method comprises ligating into a vector that encodes a portion of a gRNA a cassette comprising at least one editing cassette, a promoter, and a gene encoding another portion of the gRNA. Upon ligation, the portion of the gRNA from the editing cassette and the other portion of the gRNA are ligated and form a functional gRNA.

The present invention relates to the field of bacterial vectors and methods for genetic manipulation of bacteria. In particular, the present invention relates to a vector for genetic manipulation of a bacterium, wherein said vector comprises (a) a non-antibiotic selection marker gene cassette, (b) an origin of replication, wherein said origin of replication is not capable of inducing replication of said vector in said bacterium, and (c) a restriction endonuclease gene, a recognition site of a restriction endonuclease encoded by said restriction endonuclease gene, and a second regulatory sequence. Further, the invention relates to a bacterial host cell comprising said vector, a method for genetic manipulation of bacteria using the vector of the invention, and methods for selecting bacterial host cells.

Methods, reagents and compositions for providing more accurate and reliable genetic modification are provided. In particular a nucleic acid encoding a fusion protein comprising an endonuclease domain and a binding domain for an origin of replication is described. Also provided are methods, reagents and compositions for in vivo genetic modification of the genome of a non-animal cell or organism. Furthermore, the present application relates to uses of the said methods, reagents and compositions for introducing desirable traits to non-animal organisms or ameliorating or removing non-desirable traits in these organisms including in the treatment of disease.

The present disclosure, at least in part, provides RNA cleavage based engineered bi-stable toggle switch utilizing the Programmable Endonucleolytic Scission-Induced Stability Tuning (PERSIST) platform. Also provided herein, are vectors encoding the engineered bi-stable toggle switch, and uses thereof.

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 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 present invention provides a method of modifying a targeted site of a double stranded DNA, including a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a selected double stranded DNA and DNA glycosylase with sufficiently low reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) are bonded, with the double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into the targeted site, without cleaving at least one strand of the double stranded DNA in the targeted site.

The invention relates to vectors and methods for de-repressing Cas systems in host cells.

The invention relates to vectors and methods for de-repressing Cas systems in host cells.