Genetic targeting in non-conventional yeast using an rna-guided endonuclease

Abstract

Non-conventional yeasts are disclosed herein comprising at least one RNA-guided endonuclease (RGEN) comprising at least one RNA component that does not have a 5′-cap. This uncapped RNA component comprises a sequence complementary to a target site sequence in a chromosome or episome in the yeast. The RGEN can bind to, and optionally cleave, one or both DNA strands at the target site sequence. An example of an RGEN herein is a complex of a Cas9 protein with a guide RNA. A ribozyme is used in certain embodiments to provide an RNA component lacking a 5′-cap. Further disclosed are methods of genetic targeting in non-conventional yeast.

What is claimed is: 
     
         1 . A non-conventional yeast comprising at least one RNA-guided endonuclease (RGEN) comprising at least one RNA component that does not have a 5′-cap, wherein the RNA component comprises a sequence complementary to a target site sequence on a chromosome or episome in the yeast, wherein the RGEN can bind to the target site sequence. 
     
     
         2 . The non-conventional yeast of  claim 1 , wherein the RGEN can bind to and cleave the target site sequence. 
     
     
         3 . The non-conventional yeast of  claim 1 , wherein said yeast is a member of a genus selected from the group consisting of  Yarrowia, Pichia, Schwanniomyces, Kluyveromyces, Arxula, Trichosporon, Candida, Ustilago, Torulopsis, Zygosaccharomyces, Trigonopsis, Cryptococcus, Rhodotorula, Phaffia, Sporobolomyces , and  Pachysolen.    
     
     
         4 . The non-conventional yeast of  claim 1 , wherein the RGEN comprises a CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein-9 (Cas9) amino acid sequence. 
     
     
         5 . A non-conventional yeast comprising a Cas endonuclease and a polynucleotide sequence comprising a promoter operably linked to at least one nucleotide sequence, wherein said nucleotide sequence comprises a DNA sequence encoding a ribozyme upstream of a DNA sequence encoding an RNA component, wherein said RNA component comprises a variable targeting domain complementary to a target site sequence on a chromosome or episome in the yeast, wherein the RNA component and the Cas endonuclease can form a RNA-guided endonuclease (RGEN), wherein said RGEN can bind to the target site sequence. 
     
     
         6 . The non-conventional yeast of  claim 5 , wherein the RGEN can bind to and cleave the target site sequence. 
     
     
         7 . The non-conventional yeast of  claim 5 , wherein the RNA transcribed from the nucleotide sequence autocatalytically removes the ribozyme to yield said RNA component, wherein said RNA component does not have a 5′ cap. 
     
     
         8 . The non-conventional yeast of  claim 7 , wherein the ribozyme is a hammerhead ribozyme, hepatitis delta virus ribozyme, group I intron ribozyme, RnaseP ribozyme, or hairpin ribozyme. 
     
     
         9 . The non-conventional yeast of  claim 5 , wherein the RNA transcribed from the nucleotide sequence does not autocatalytically removes the ribozyme to yield a ribozyme-RNA component fusion molecule without a 5′ cap. 
     
     
         10 . A method for modifying a target site on a chromosome or episome in a non-conventional yeast, the method comprising providing to a non-conventional yeast a first recombinant DNA construct comprising a DNA sequence encoding a Cas endonuclease, and a second recombinant DNA construct comprising a DNA sequence encoding a ribozyme upstream of an RNA component, wherein the RNA transcribed from the second recombinant DNA construct autocatalytically removes the ribozyme to yield said RNA component, wherein the Cas9 endonuclease introduces a single or double-strand break at said target site. 
     
     
         11 . A method for modifying a target site on a chromosome or episome in a non-conventional yeast, the method comprising providing to a non-conventional yeast a first recombinant DNA construct comprising a DNA sequence encoding a Cas endonuclease, and a second recombinant DNA construct comprising a DNA sequence encoding a ribozyme-RNA component fusion molecule, wherein said ribozyme-RNA component fusion molecule and Cas9 endonuclease can form a RGEN that introduces a single or double-strand break at said target site. 
     
     
         12 . The method of any of  claims 10 - 11 , further comprising identifying at least one non-conventional yeast cell that has a modification at said target, wherein the modification includes at least one deletion, addition or substitution of one or more nucleotides in said target site. 
     
     
         13 . The method of any of  claims 10 - 11 , further comprising providing a donor DNA to said yeast, wherein said donor DNA comprises a polynucleotide of interest. 
     
     
         14 . The method of  claim 13 , further comprising identifying at least one yeast cell comprising in its chromosome or episome the polynucleotide of interest integrated at said target site. 
     
     
         15 . A method for editing a nucleotide sequence on a chromosome or episome in a non-conventional yeast, the method comprising providing to a non-conventional yeast a polynucleotide modification template DNA, a first recombinant DNA construct comprising a DNA sequence encoding a Cas endonuclease, and a second recombinant DNA construct comprising a DNA sequence encoding a ribozyme upstream of an RNA component, wherein the RNA transcribed from the second recombinant DNA construct autocatalytically removes the ribozyme to yield said RNA component, wherein the Cas9 endonuclease introduces a single or double-strand break at a target site in the chromosome or episome of said yeast, wherein said polynucleotide modification template DNA comprises at least one nucleotide modification of said nucleotide sequence. 
     
     
         16 . A method for silencing a nucleotide sequence on a chromosome or episome in a non-conventional yeast, the method comprising providing to a non-conventional yeast, at least a first recombinant DNA construct comprising a DNA sequence encoding an inactivated Cas9 endonuclease, and at least a second recombinant DNA construct comprising a promoter operably linked to at least one polynucleotide, wherein said at least one polynucleotide encodes a ribozyme-RNA component fusion molecule, wherein said ribozyme-RNA component fusion molecule and the inactivated Cas9 endonuclease can form a RGEN that binds to said nucleotide sequence in the chromosome or episome of said yeast, thereby blocking transcription of said nucleotide sequence. 
     
     
         17 . A high throughput method for the production of multiple guide RNAs for gene modification in non-conventional yeast, the method comprising:
 a) providing a recombinant DNA construct comprising a promoter operably linked to, in 5′ to 3′ order, a first DNA sequence encoding a ribozyme, a second DNA sequence encoding a counterselection agent, a third DNA sequence encoding a CER domain of a guide RNA, and a terminator sequence;   b) providing at least one oligonucleotide duplex to the recombinant DNA construct of (a), wherein said oligonucleotide duplex is originated from combining a first single stranded oligonucleotide comprising a DNA sequence capable of encoding a variable targeting domain (VT) of a guide RNA target sequence with a second single stranded oligonucleotide comprising the complementary sequence to the DNA sequence encoding the variable targeting domain;   c) exchanging the counterselection agent of (a) with the at least one oligoduplex of (b), thereby creating a library of recombinant DNA constructs each comprising a DNA sequence capable of encoding a variable targeting domain of a guide RNA; and,   d) transcribing the library of recombinant DNA constructs of (c), thereby creating a library of ribozyme-guideRNA molecules.   
     
     
         18 . The method of  claim 17 , further comprising inducing the library of ribozyme-guide RNA molecules such that said molecules autocatalitically remove the ribozyme and any RNA sequence upstream of the ribozyme to yield a library of guide RNA molecules that do not contain 5′ cap. 
     
     
         19 . The method of  claim 17 , further comprising inducing the library of ribozyme-guide RNA molecules such that said molecules cleaves any RNA sequence upstream of the ribozyme to yield a ribozyme-gRNA fusion molecules that do not contain 5′ cap. 
     
     
         20 . A recombinant DNA sequence comprising (i) a polymerase-II promoter operably linked to (ii) a nucleotide sequence encoding a ribozyme and a guide RNA, wherein said ribozyme is upstream of said guide RNA, wherein RNA transcribed from the nucleotide sequence of (ii) autocatalically removes the ribozyme to yield said guide RNA, and wherein said guide RNA can form a RGEN that can recognize, bind to, and optionally cleave a target site in the genome of a non-conventional yeast.