Method to produce a circular vector comprising two or more guide-polynucleotide expression cassettes, wherein said two or more guide-polynucleotide expression cassettes comprise 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 steps: - providing two or more polynucleotides, wherein the two or more polynucleotides comprise a polynucleotide sequence to be extended, wherein the polynucleotide sequence to be extended comprises at the 5'-end and/or at the 3'-end to be extended a fragment of a guide-polynucleotide expression cassette, wherein said fragment of a guide-polynucleotide expression cassette at the 5'-end and/or at the 3'-end of the polynucleotide sequence to be extended, does not comprise a polynucleotide coding for a guide sequence; - performing two or more overlap-extension PCR reactions by subjecting in each reaction one of said two or more polynucleotides and two suitable polynucleotide primers wherein at least one suitable primer comprises a polynucleotide coding for a guide sequence to yield one hybrid linear polynucleotide, wherein the two or more polynucleotide sequence to be extended and the suitable polynucleotide primers are selected so that each hybrid linear polynucleotide obtained in the two or more overlap-extension PCR reactions comprises at least a polynucleotide coding for a guide sequence and suitable 5'-termini and 3'-termini which allow assembly in vivo of the two or more hybrid linear polynucleotides and optionally of one or more additional linear polynucleotides with each other in a pre-defined order to yield a circular vector comprising two or more functional guide-polynucleotide expression cassettes; - subjecting the two or more hybrid linear polynucleotides obtained in the overlap-extension PCR and optionally one or more additional linear polynucleotides to an assembly reaction yielding a circular vector comprising one or more functional guide-polynucleotide expression cassettes, wherein the assembly reaction occurs in vivo, preferably wherein the guide-polynucleotide is a gRNA. - providing two or more polynucleotides, wherein the two or more polynucleotides comprise a polynucleotide sequence to be extended, wherein the polynucleotide sequence to be extended comprises at the 5'-end and/or at the 3'-end to be extended a fragment of a guide-polynucleotide expression cassette, wherein said fragment of a guide-polynucleotide expression cassette at the 5'-end and/or at the 3'-end of the polynucleotide sequence to be extended, does not comprise a polynucleotide coding for a guide sequence; - performing two or more overlap-extension PCR reactions by subjecting in each reaction one of said two or more polynucleotides and two suitable polynucleotide primers wherein at least one suitable primer comprises a polynucleotide coding for a guide sequence to yield one hybrid linear polynucleotide, wherein the two or more polynucleotide sequence to be extended and the suitable polynucleotide primers are selected so that each hybrid linear polynucleotide obtained in the two or more overlap-extension PCR reactions comprises at least a polynucleotide coding for a guide sequence and suitable 5'-termini and 3'-termini which allow assembly in vivo of the two or more hybrid linear polynucleotides and optionally of one or more additional linear polynucleotides with each other in a pre-defined order to yield a circular vector comprising two or more functional guide-polynucleotide expression cassettes; - subjecting the two or more hybrid linear polynucleotides obtained in the overlap-extension PCR and optionally one or more additional linear polynucleotides to an assembly reaction yielding a circular vector comprising one or more functional guide-polynucleotide expression cassettes, wherein the assembly reaction occurs in vivo, preferably wherein the guide-polynucleotide is a gRNA.