Avoiding epigenetic silencing of exogenous nucleic acid in algae
The present application relates to the identification of novel DNA methyltransferases including CHG methylation in algal species. The present application relates to algal mutants permitting the expression of exogenous genes by alleviating the epigenetic mechanisms of CHG and CHH methylation of exogenous DNA and mono- and tri-methylation of lysine 9 of histone 3 (H3K9). This is achieved by mutating or attenuating the methyltransferase (MTase) genes in algae. The present application also relates to methods for efficiently expressing exogenous genes in algal species.
1. A mutant photosynthetic organism comprising a mutated or attenuated gene encoding a polypeptide having a CHG DNA methyltransferase activity, wherein the mutation or attenuation is in the gene that encodes the polypeptide comprising an amino acid sequence having at least 80% identity to an amino acid sequence of any of SEQ ID NO: 1, 3, 5, 7 or 28, and
wherein the mutant photosynthetic microorganism has reduced CHG DNA methylation as compared to a control photosynthetic organism without a mutated or attenuated gene encoding a polypeptide having a CHG DNA methyltransferase activity.
2. The mutant photosynthetic organism of claim 1 , wherein the mutant is a genetically engineered mutant.
3. The mutant photosynthetic organism of claim 2 , wherein the mutant has been genetically engineered by insertional mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome engineering, one or more ribozymes, and/or a CRISPR/Cas system.
4. The mutant photosynthetic organism of claim 3 , wherein the mutant has been genetically engineered by a CRISPR/Cas system.
5. The mutant photosynthetic organism of claim 1 , wherein the mutant has been generated by UV irradiation, gamma irradiation, or chemical mutagenesis.
6. The mutant photosynthetic organism of claim 1 , wherein the polypeptide having a CHG DNA methyltransferase activity comprises an amino acid sequence having at least 80% identity to an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28.
7. The mutant photosynthetic organism of claim 1 , wherein the mutant photosynthetic organism further comprises an exogenous DNA, and wherein the reduced CHG DNA methylation is in the exogenous DNA.
8. The mutant photosynthetic organism of claim 7 , wherein the exogenous DNA is integrated into the genome of the photosynthetic organism.
9. The mutant photosynthetic organism of claim 1 , wherein the reduced CHG DNA methylation is in a DNA sequence native to the photosynthetic organism.
10. The mutant photosynthetic organism of claim 9 , wherein the wherein the reduced CHG DNA methylation is in the centromere or highly repetitive DNA regions of the mutant photosynthetic organism.
11. The mutant photosynthetic organism of claim 7 , wherein the expression of the exogenous nucleic acid is greater as compared to a control photosynthetic organism comprising the exogenous nucleic acid but without a mutated or attenuated gene encoding a polypeptide having a CHG DNA methyltransferase activity.
12. The mutant photosynthetic organism according to claim 1 , wherein the mutant photosynthetic organism has reduced monomethylation or trimethylation of lysine 9 of histone H3 (H3K9).
13. The mutant photosynthetic organism according to claim 1 , wherein the mutant photosynthetic organism has reduced CHH DNA methylation as compared to a control photosynthetic organism without a mutated or attenuated gene encoding a polypeptide having a CHG DNA methyltransferase activity.
14. The mutant photosynthetic organism according to claim 1 , wherein the photosynthetic organism is algae.
15. A method of enhancing the expression of an exogenous DNA in a photosynthetic organism, comprising:
a) introducing an exogenous DNA into the photosynthetic organism;
b) mutating or attenuating a gene encoding a polypeptide having a CHG DNA methyltransferase activity, wherein the mutation or attenuation is in the gene that encodes the polypeptide comprising an amino acid sequence having at least 80% identity to an amino acid sequence of any of SEQ ID NO: 1, 3, 5, 7 or 28,
wherein the mutant photosynthetic organism has reduced CHG DNA methylation of the exogenous DNA as compared to a control photosynthetic organism comprising the exogenous DNA but without a mutated or attenuated gene encoding a polypeptide having a CHG DNA methyltransferase activity; and
wherein the expression of the exogenous DNA is enhanced in the photosynthetic organism as compared to the control photosynthetic organism.
16. The method of claim 15 , wherein the mutating or attenuating of the gene encoding a polypeptide having a CHG DNA methyltransferase activity is by genetic engineering.
17. The method of claim 16 , wherein the genetically engineering is by insertional mutagenesis, gene replacement, RNAi, antisense RNA, meganuclease genome engineering, one or more ribozymes, and/or a CRISPR/Cas system.
18. The method of claim 17 , wherein the genetically engineering is by a CRISPR/Cas system.
19. The method of claim 15 , wherein the mutating or attenuating of the gene encoding a polypeptide having a CHG DNA methyltransferase activity is by UV irradiation, gamma irradiation, or chemical mutagenesis.
20. The method of claim 15 , wherein the polypeptide having a CHG DNA methyltransferase activity comprises an amino acid sequence having at least 80% identity to an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28.
21. The method of claim 15 , wherein the mutant photosynthetic organism has reduced CHH DNA methylation as compared to a control photosynthetic organism without a mutated or attenuated gene encoding a polypeptide having a CHG DNA methyltransferase activity.
22. The method of claim 15 , wherein the exogenous DNA is integrated into the genome of the photosynthetic organism.
23. The method of claim 15 , wherein the photosynthetic organism is algae.