CRISPR-CAS system for a lipolytic yeast host cell

A non-naturally occurring or engineered composition comprising a CRISPR-Cas system comprising guide-polynucleotide and a Cas protein, wherein the guide-polynucleotide comprises a guide-sequence that is the reverse complement of a target-polynucleotide sequence in a host cell, which host cell is a Yarrowia lipolytic yeast, wherein the guide-polynucleotide directs binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex, wherein the guide-polynucleotide sequence is 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 PAM is 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;

• and W is A or T, wherein the guide-polynucleotide is encoded by a polynucleotide that is operably linked to an RNA polymerase II promoter, wherein the polynucleotide that is operably linked to an RNA polymerase II promoter further encodes a pre-guide polynucleotide comprising the guide-polynucleotide and at least one self-processing ribozyme, wherein, when transcribed, the guide-polynucleotide is released by the self-processing ribozyme from the pre-guide-polynucleotide transcript.