RNA-Guided Genome Editing for Target Gene Mutations in Wheat

Abstract

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas–mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3′ end of the target site abolished the cleavage activity completely. The mismatches at the 5′ end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3′ end. This approach provides a powerful method for genome engineering in plants.

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References 18

1. 1.

   Brouns S. J. Jore M. M. Lundgren M. Westra E. R. Slijkhuis R. J., 2008 Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321: 960–964.

   Not in our library10.1126/science.1159689

2. 2.

   Bos J. I. B. Kanneganti T. D. Young C. Cakir C. Huitema E., 2006 The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant J. 48: 165–176.

   Not in our library10.1111/j.1365-313x.2006.02866.x

3. 3.

   Chen K. Gao C., 2013 TALENs: Customizable molecular DNA scissors for genome engineering of plants. J. Genet. Genomics 40: 271–279.

   Not in our library10.1016/j.jgg.2013.03.009

4. 4.

   Chylinski K. Le R. A. Charpentier E., 2013 The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol. 10: 1–12.

   Not in our library10.4161/rna.24321

5. 5.

   Cong L. Ran F. A. Cox D. Lin S. Barretto R., 2013 Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823.

   Not in our library10.1126/science.1231143

6. 6.

   DiCarlo J. E. Norville J. E. Mali P. Rios X. Aach J., 2013 Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41: 4336–4343.

   Not in our library10.1093/nar/gkt135

7. 7.

   Fu Y. Foden J. A. Khayter C. Maeder M. L. Reyon D., 2013 High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31: 822–826. DOI: 10.1038/nbt.2623.

   Not in our library10.1038/nbt.2623

8. 8.

   Gasiunas G. Barrangou R. Horvath P. Siksnys V., 2012 Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. U.S.A. 109. 109: E2579–E2586.

   Not in our library10.1073/pnas.1208507109

9. 9.

   Hale C. R. Zhao P. Olson S. Duff M. O. Graveley B. R., 2009 RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139: 945–956.

   Not in our library10.1016/j.cell.2009.07.040

10. 10.

    Hwang W. Y. Fu Y. Reyon D. Maeder M. L. Tsai S. Q., 2013 Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31: 227–229.

    Not in our library10.1038/nbt.2501

11. 11.
    RNA-programmed genome editing in human cells · 2013

    No notice
12. 12.

    Jore M. M. Lundgren M. Duijn E. V. Bultema J. B. Westra E. R., 2011 Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat. Struct. Mol. Biol. 18: 529–536.

    Not in our library10.1038/nsmb.2019

13. 13.

    Li J. F. Norville J. E. Aach J. McCormack M. Zhang D., 2013 Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31: 688–691. DOI: 10.1038/nbt.2654.

    Not in our library10.1038/nbt.2654

14. 14.

    Mali P. Yang L. Esvelt K. M. Aach J. Guell M., 2013 RNA-guided human genome engineering via Cas9. Science 339: 823–826.

    Not in our library10.1126/science.1232033

15. 15.

    Nekrasov V. Staskawicz B. Weigel D. Jones J. D. J. Kamoun S., 2013 Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31: 691–693. DOI: 10.1038/nbt.2655.

    Not in our library10.1038/nbt.2655

16. 16.

    Shan Q. Wang Y. Li J. Zhang Y. Chen K., 2013 Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31: 686–688. DOI: 10.1038/nbt.2650.

    Not in our library10.1038/nbt.2650

17. 17.

    Wu H. McCormac A. C. Elliott M. C. Chen D. F., 1998 Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare). Plant Cell Tissue Organ Cult. 54: 161–171.

    Not in our library

18. 18.

    Zhang F. Maeder M. L. Unger-Wallace E. Hoshaw J. P. Reyon D., 2010 High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc. Natl. Acad. Sci. USA 107: 12028–12033.

    Not in our library10.1073/pnas.0914991107

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