Crispr-cas system for an algal host cell

Abstract

The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-Cas system for a Labyrinthulomycetes host cell.

1 . A non-naturally occurring or engineered composition comprising a source of a CRISPR-Cas system comprising a guide-polynucleotide and a Cas protein, wherein the guide-polynucleotide comprises a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and the guide-polynucleotide 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 selected from the group consisting of  Aurantiochytrium, Oblongichytrium, Schizochytrium, Thraustochytrium,  and  Ulkenia , and 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 or analog thereof; and W is A or T. 
     
     
         2 . The composition according to  claim 1 , wherein the Cas protein is encoded by a polynucleotide and the guide-polynucleotide is encoded by or present on a polynucleotide. 
     
     
         3 . The composition according to  claim 2 , wherein each of the polynucleotides are comprised in a vector. 
     
     
         4 . The composition according to  claim 1 , wherein the guide polynucleotide is encoded by a polynucleotide that is transcribed to provide for the actual guide-polynucleotide. 
     
     
         5 . The composition according to  claim 3 , wherein the polynucleotides are comprised in one vector. 
     
     
         6 . The composition according to  claim 3 , wherein the vector encoding the Cas protein is driven by a low strength promoter and the vector encoding the guide-polynucleotide is driven by a high strength promoter. 
     
     
         7 . The composition according to  claim 6 , wherein the vectors comprise distinct selectable markers 
     
     
         8 . The composition according to  claim 2 , wherein the polynucleotide encoding the Cas protein comprises at least one nuclear localization sequence, preferably a heterologous nuclear localization sequence. 
     
     
         9 . The composition according to  claim 1 , wherein the Cas protein has activity for directing cleavage of both polynucleotide strands at the location of the target-sequence. 
     
     
         10 . The composition according to  claim 2 , wherein the polynucleotide encoding the Cas protein is codon optimized for the host cell. 
     
     
         11 . The composition according to  claim 2 , wherein the polynucleotide encoding the guide-polynucleotide is operably linked to a RNA polymerase II or II I promoter, preferably to a Labyrinthulomycete EF-1 promoter. 
     
     
         12 . The composition according to  claim 2 , wherein the polynucleotide encoding the guide-polynucleotide comprises an RNA polymerase II promoter and self-processing ribozymes, and wherein, when transcribed, the guide-polynucleotide is released by the self-processing ribozymes from the pre-guide-polynucleotide transcript. 
     
     
         13 . A method of modulating expression of a polynucleotide in a cell, comprising contacting a host cell with the composition according to  claim 1 , wherein the guide-polynucleotide directs binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex. 
     
     
         14 . The method according to  claim 13 , wherein the host cell comprises a polynucleotide encoding a compound of interest. 
     
     
         15 . The method according to  claim 14 , wherein the host cell is a recombinant host cell. 
     
     
         16 . A host cell comprising the composition according to  claim 1 . 
     
     
         17 . A method for the production of a compound of interest, comprising culturing under conditions conducive to the production of the compound of interest a host cell according to  claim 16  and recovering the compound of interest.