The present invention generally relates to improved methods of assembly of two or more DNA fragments, methods of rapid ligation-independent cloning, and kits for rapid ligation-independent cloning and their uses.

The present invention generally relates to improved methods of assembly of two or more DNA fragments, methods of rapid ligation-independent cloning, and kits for rapid ligation-independent cloning and their uses.

The disclosure discloses an in-vivo continuous directed evolution system and application thereof, and belongs to the fields of gene engineering and enzyme engineering. The system includes Escherichia coli host bacteria carrying a random mutation module mutagenesis plasmid, a programmed death module toxin-antitoxin system and a target gene expression module target plasmid. The modules are coupled with one another, and target genes are subjected to multiple rounds of continuous mutation by virtue of the random mutation module mutagenesis plasmid in the system, so that the mutation rate of the target genes is further increased, and ultimately, efficient evolution and screening of the target genes in the host bacteria are realized. According to the system, mutations are accurately positioned on the target genes, random mutations in non-target gene regions are reduced, and the system has good practical value and can be applied to directed evolution of various different functional proteins.

The disclosure discloses an in-vivo continuous directed evolution system and application thereof, and belongs to the fields of gene engineering and enzyme engineering. The system includes Escherichia coli host bacteria carrying a random mutation module mutagenesis plasmid, a programmed death module toxin-antitoxin system and a target gene expression module target plasmid. The modules are coupled with one another, and target genes are subjected to multiple rounds of continuous mutation by virtue of the random mutation module mutagenesis plasmid in the system, so that the mutation rate of the target genes is further increased, and ultimately, efficient evolution and screening of the target genes in the host bacteria are realized. According to the system, mutations are accurately positioned on the target genes, random mutations in non-target gene regions are reduced, and the system has good practical value and can be applied to directed evolution of various different functional proteins.

Methods are provided herein for assembling at least two nucleic acids using a sequence specific nuclease agent (e.g., a gRNA-Cas complex) to create end sequences having complementarity and subsequently assembling the overlapping complementary sequences. The nuclease agent (e.g., a gRNA-Cas complex) can create double strand breaks in dsDNA in order to create overlapping end sequences or can create nicks on each strand to produce complementary overhanging end sequences. Assembly using the method described herein can assemble any nucleic acids having overlapping sequences or can use a joiner oligo to assemble sequences without complementary ends.

Methods are provided herein for assembling at least two nucleic acids using a sequence specific nuclease agent (e.g., a gRNA-Cas complex) to create end sequences having complementarity and subsequently assembling the overlapping complementary sequences. The nuclease agent (e.g., a gRNA-Cas complex) can create double strand breaks in dsDNA in order to create overlapping end sequences or can create nicks on each strand to produce complementary overhanging end sequences. Assembly using the method described herein can assemble any nucleic acids having overlapping sequences or can use a joiner oligo to assemble sequences without complementary ends.

This document provides methods and materials for assembling nucleic acid constructs (e.g., TALENs). For example, methods for assembling TALEs that are rapid, flexible for use in many cloning scaffolds (such as common nuclease and nickase backbones), and achievable with standard molecular biology laboratory tools, thereby making TALEs a more accessible genome system, are provided.

Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.

Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.

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.