Guide rna assembly vector

A method of assembling a circular vector comprising one or more guide-polynucleotide expression cassettes in vivo, wherein said one or more guide-polynucleotide expression cassette comprises a polynucleotide encoding a guide-polynucleotide operably linked to one or more control sequences which direct the expression of said guide-polynucleotide in a host cell, wherein said guide-polynucleotide comprises a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and wherein the guide-polynucleotide can direct binding of a Cas protein at a target-polynucleotide in a host cell to form a CRISPR-Cas complex, wherein the method comprises the following:

• providing one or more linear polynucleotides, wherein said one or more linear polynucleotides comprise at the 5′-terminus and/or at the 3′-terminus at least one guide-polynucleotide expression cassette or a fragment of at least one guide-polynucleotide expression cassette comprising at least the guide sequence;
• transforming said one or more linear polynucleotides, and optionally one or more additional linear polynucleotides into a host cell;
• allowing an homologous recombination reaction to take place between said one or more linear polynucleotides and optionally one or more additional linear polynucleotides, wherein said linear polynucleotides and additional linear polynucleotides have been selected to comprise suitable 5′-termini and 3′-termini which allow homologous recombination of said one or more linear polynucleotides and optionally of said one or more additional linear polynucleotides with each other in a pre-defined order to yield a circular vector comprising one or more functional guide-polynucleotide expression cassettes;
• optionally selecting a host cell comprising a circular vector and wherein said vector comprises one or more functional guide-polynucleotide expression cassettes;
• optionally recovering the circular vector.

A method for producing a recombinant host cell comprising a circular vector, wherein said vector comprises one or more guide-polynucleotide expression cassettes, wherein said one or more guide-polynucleotide expression cassette comprises a polynucleotide encoding a guide-polynucleotide operably linked to one or more control sequences which direct the expression of said guide-polynucleotide in a host cell, wherein said guide-polynucleotide comprises a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and wherein the guide-polynucleotide can direct binding of a Cas protein at a target-polynucleotide in a host cell to form a CRISPR-Cas complex, wherein the method comprises the following:

• providing one or more linear polynucleotides, wherein said one or more linear polynucleotides comprise at the 5′-terminus and/or at the 3′-terminus at least one guide-polynucleotide expression cassette or a fragment of at least one guide-polynucleotide expression cassette comprising at least the guide sequence;
• transforming said one or more linear polynucleotides, and optionally one or more additional linear polynucleotides into a host cell;
• allowing an homologous recombination reaction to take place between said one or more linear polynucleotides and optionally one or more additional linear polynucleotides, wherein said linear polynucleotides and additional linear polynucleotides have been selected to comprise suitable 5′-termini and 3′-termini which allow homologous recombination of said one or more linear polynucleotides and optionally of said one or more additional linear polynucleotides with each other in a pre-defined order to yield a circular vector comprising one or more functional guide-polynucleotide expression cassettes;
• optionally selecting a host cell comprising a circular vector and wherein said vector comprises one or more functional guide-polynucleotide expression cassettes.