A non-naturally occurring or engineered composition comprising: - a vector comprising a selectable marker, and a source of a CRISPR-Cas system comprising a Cas protein and at least two distinct polynucleotides each encoding a respective distinct guide-polynucleotide, wherein the guide-polynucleotides comprise a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and the guide-polynucleotides can direct binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex, wherein the guide-sequence is essentially the reverse complement of the (N)y part of a 5'-(N)yPAM-3' polynucleotide sequence target in the genome of the host cell, wherein y is an integer of 8-30, wherein PAM is a protospacer adjacent motif, wherein the host cell is a eukaryote, which eukaryote is a yeast selected from a Saccharomyces or a Kluyveromyces, wherein PAM is preferably a sequence selected from the group consisting of 5'-XGG-3', 5'-XGGXG-3', 5'-XXAGAAW-3', 5'-XXXXGATT-3', 5'-XXAGAA-3', 5'-XAAAAC-3', wherein X can be any nucleotide or analog thereof, preferably X can be any nucleotide; and W is A or T, wherein the at least two distinct polynucleotides each encoding a respective distinct guide-polynucleotide have regions of sequence identity with the vector such that homologous recombination of said polynucleotides and the vector is facilitated, wherein the vector is preferably linear, and wherein the at least two distinct polynucleotides each encoding a respective distinct guide-polynucleotide additionally comprise sequence identity with each other such that recombination of said polynucleotides with each other and with said vector is facilitated, wherein the recombination is in vivo recombination in the host cell. - a vector comprising a selectable marker, and a source of a CRISPR-Cas system comprising a Cas protein and at least two distinct polynucleotides each encoding a respective distinct guide-polynucleotide, wherein the guide-polynucleotides comprise a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and the guide-polynucleotides can direct binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex, wherein the guide-sequence is essentially the reverse complement of the (N)y part of a 5'-(N)yPAM-3' polynucleotide sequence target in the genome of the host cell, wherein y is an integer of 8-30, wherein PAM is a protospacer adjacent motif, wherein the host cell is a eukaryote, which eukaryote is a yeast selected from a Saccharomyces or a Kluyveromyces, wherein PAM is preferably a sequence selected from the group consisting of 5'-XGG-3', 5'-XGGXG-3', 5'-XXAGAAW-3', 5'-XXXXGATT-3', 5'-XXAGAA-3', 5'-XAAAAC-3', wherein X can be any nucleotide or analog thereof, preferably X can be any nucleotide; and W is A or T, wherein the at least two distinct polynucleotides each encoding a respective distinct guide-polynucleotide have regions of sequence identity with the vector such that homologous recombination of said polynucleotides and the vector is facilitated, wherein the vector is preferably linear, and wherein the at least two distinct polynucleotides each encoding a respective distinct guide-polynucleotide additionally comprise sequence identity with each other such that recombination of said polynucleotides with each other and with said vector is facilitated, wherein the recombination is in vivo recombination in the host cell. <EOS>