Compositions and methods for helper strain-mediated fungal genome modification

Patent metadata

Field	Value
Publication number	US-10655148-B2
Application number	US-201515536892-A
Country	US
Kind code	B2
Filing date	Dec 16, 2015
Priority date	Dec 16, 2014
Publication date	May 19, 2020
Grant date	May 19, 2020

Abstract

Compositions and methods are provided employing a helper strain system for promoting genetic alterations in a fungal host cell, e.g., a filamentous fungal host cell.

First claim

That which is claimed:

1. A method for homologous recombination of a donor DNA with a genomic locus in a Trichoderma sp. cell; the method comprising:
(a) generating a heterokaryon between a helper Trichoderma species (sp.) strain and a target Trichoderma sp, strain, wherein the helper Trichoderma sp. strain comprises an expression construct that silences the non-homologous end joining (NHEJ) mechanism, wherein the expression construct silences one or more of: ku80, ku70 and liq 4:
(b) introducing a donor DNA into the heterokaryon, wherein the donor DNA comprises a region of homology to a genomic locus in the target strain sufficient for homologous recombination at the genomic locus;
(c) generating and plating spores from the heterokaryon cells of (b); and,
(d) identifying cells from the plated spores in which (i) the donor DNA has integrated into the genome by homologous recombination at the genomic locus, and (ii) the expression construct that silences the non-homologous end joining (NHEJ) mechanism is not present.

2. The method of claim 1 , further comprising introducing a functional Cas/guide RNA complex into the heterokaryon, wherein the Cas/guide RNA complex has a target site within the genomic locus.

3. The method of claim 2 , wherein the Cas is a Cas nickase.

4. The method of claim 1 , wherein the donor DNA comprises a polynucleotide sequence of interest, wherein homologous recombination at the genomic locus results in insertion of the polynucleotide sequence of interest in the genomic locus.

5. The method of claim 2 , wherein the Cas is a Cas9 endonuclease or variant thereof.

6. The method of claim 5 , wherein the Cas9 endonuclease or variant thereof comprises a full length Cas9 or a functional fragment thereof from a cell type selected from the group consisting of: Streptococcus sp., S. pyogenes, S. mutans, S. thermophiles, Campylobacter sp., C. jejuni, Neisseria sp., N. meningitides, Francisella sp., F. novicida, Pasteurella sp., and P. multocida.

7. The method of claim 1 , wherein introducing the functional Cas/guide RNA complex into the heterokaryon comprises introducing a DNA construct comprising an expression cassette for the Cas endonuclease into the Trichoderma sp. cells.

8. The method of claim 1 , wherein introducing the functional Cas/guide RNA complex into the heterokaryon comprises introducing a DNA construct comprising an expression cassette for the guide RNA into the Trichoderma sp. cells.

9. The method of claim 1 , wherein the introducing step comprises directly introducing the Cas endonuclease into the Trichoderma sp. cells.

10. The method of claim 1 , wherein the introducing step comprises directly introducing the guide RNA into the Trichoderma sp. cells.

11. The method of claim 1 , wherein the Trichoderma sp. is Trichoderma reesei.

12. The method of claim 1 , wherein the donor DNA has partially integrated into the genome at the genomic locus.

13. The method of claim 1 , wherein integration of the donor DNA results in a modification of the genomic locus.

14. The method of claim 13 , wherein the modification is selected from the group consisting of a deletion of one or more nucleotides, an insertion of one or more nucleotides, insertion of an expression cassette encoding a protein of interest, a substitution of one or more nucleotides, and any combination thereof.

15. The method of claim 1 , wherein the identifying step comprises culturing cells grown from the spores from step (c) under conditions to select for or screen for the integration of the donor DNA at the genomic locus or the modification of the genomic locus.

Assignees

• Danisco Us Inc

Inventors

• Virag Aleksandra
• Ward Michael

Classifications

• C12N15/902 Primary

  using homologous recombination · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/80

  for fungi · CPC title

• C12N15/04

  Fungi · CPC title

• C12N2510/02

  Cells for production · CPC title