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RNA-guided genome editing for target gene mutations in wheat.

Upadhyay SK, Kumar J, Alok A, Tuli R - G3 (Bethesda) (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: National Agri-Food Biotechnology Institute, Department of Biotechnology, Government of India, Mohali, Punjab, India 160071.

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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Basic architecture of constructs used for CRISPR-Cas–mediated genome editing and expression analysis of cgRNA and Cas9. (A) Architecture of constructs used for expression. The four types of constructs developed for the expression of cgRNA and Cas9 are shown. (B) RT-PCR analysis of expression of cgRNA and Cas9 in wheat: untransformed (lane 1); pCinox1-transformed (lane 2) suspension cells; and pCwpds1-transformed (lane 3) suspension cells RT-PCR analysis of expression of cgRNA and Cas9 in. N. benthamiana: untransformed (lane 4); pTpds1 (lane 5); pCas9-transformed (lane 6) leaf; and pCtpds1-transformed (lane 7) leaf. (C) Western blot analysis of Cas9 using anti-Flag antibody (FLAG tag attached at N-terminus of Cas9). Lane M, molecular weight marker; lane 1, untransformed; lane 2, pCinox1-transformed suspension cells of wheat; lane 3, pCwpds1-transformed suspension cells of wheat; lane 4, pCtpds1 agro-infiltrated; lane 5, untransformed N. benthamiana leaves.
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fig1: Basic architecture of constructs used for CRISPR-Cas–mediated genome editing and expression analysis of cgRNA and Cas9. (A) Architecture of constructs used for expression. The four types of constructs developed for the expression of cgRNA and Cas9 are shown. (B) RT-PCR analysis of expression of cgRNA and Cas9 in wheat: untransformed (lane 1); pCinox1-transformed (lane 2) suspension cells; and pCwpds1-transformed (lane 3) suspension cells RT-PCR analysis of expression of cgRNA and Cas9 in. N. benthamiana: untransformed (lane 4); pTpds1 (lane 5); pCas9-transformed (lane 6) leaf; and pCtpds1-transformed (lane 7) leaf. (C) Western blot analysis of Cas9 using anti-Flag antibody (FLAG tag attached at N-terminus of Cas9). Lane M, molecular weight marker; lane 1, untransformed; lane 2, pCinox1-transformed suspension cells of wheat; lane 3, pCwpds1-transformed suspension cells of wheat; lane 4, pCtpds1 agro-infiltrated; lane 5, untransformed N. benthamiana leaves.

Mentions: Four types of expression vectors were prepared by cloning cgRNA and Cas9 in different combinations (Figure 1A). Constitutive CaMVE35S promoter was used to drive the expression. We cloned cgRNA and Cas9 alone, as well as together, in plant expression vector pBI121. The Cas9 gene (4272 bp) including the FLAG tag and nuclear localization signal for eukaryotic expression was obtained from Addgene USA (Addgene plasmid 42229). The plant expression constructs and their target genes are provided in Table S1.


RNA-guided genome editing for target gene mutations in wheat.

Upadhyay SK, Kumar J, Alok A, Tuli R - G3 (Bethesda) (2013)

Basic architecture of constructs used for CRISPR-Cas–mediated genome editing and expression analysis of cgRNA and Cas9. (A) Architecture of constructs used for expression. The four types of constructs developed for the expression of cgRNA and Cas9 are shown. (B) RT-PCR analysis of expression of cgRNA and Cas9 in wheat: untransformed (lane 1); pCinox1-transformed (lane 2) suspension cells; and pCwpds1-transformed (lane 3) suspension cells RT-PCR analysis of expression of cgRNA and Cas9 in. N. benthamiana: untransformed (lane 4); pTpds1 (lane 5); pCas9-transformed (lane 6) leaf; and pCtpds1-transformed (lane 7) leaf. (C) Western blot analysis of Cas9 using anti-Flag antibody (FLAG tag attached at N-terminus of Cas9). Lane M, molecular weight marker; lane 1, untransformed; lane 2, pCinox1-transformed suspension cells of wheat; lane 3, pCwpds1-transformed suspension cells of wheat; lane 4, pCtpds1 agro-infiltrated; lane 5, untransformed N. benthamiana leaves.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852385&req=5

fig1: Basic architecture of constructs used for CRISPR-Cas–mediated genome editing and expression analysis of cgRNA and Cas9. (A) Architecture of constructs used for expression. The four types of constructs developed for the expression of cgRNA and Cas9 are shown. (B) RT-PCR analysis of expression of cgRNA and Cas9 in wheat: untransformed (lane 1); pCinox1-transformed (lane 2) suspension cells; and pCwpds1-transformed (lane 3) suspension cells RT-PCR analysis of expression of cgRNA and Cas9 in. N. benthamiana: untransformed (lane 4); pTpds1 (lane 5); pCas9-transformed (lane 6) leaf; and pCtpds1-transformed (lane 7) leaf. (C) Western blot analysis of Cas9 using anti-Flag antibody (FLAG tag attached at N-terminus of Cas9). Lane M, molecular weight marker; lane 1, untransformed; lane 2, pCinox1-transformed suspension cells of wheat; lane 3, pCwpds1-transformed suspension cells of wheat; lane 4, pCtpds1 agro-infiltrated; lane 5, untransformed N. benthamiana leaves.
Mentions: Four types of expression vectors were prepared by cloning cgRNA and Cas9 in different combinations (Figure 1A). Constitutive CaMVE35S promoter was used to drive the expression. We cloned cgRNA and Cas9 alone, as well as together, in plant expression vector pBI121. The Cas9 gene (4272 bp) including the FLAG tag and nuclear localization signal for eukaryotic expression was obtained from Addgene USA (Addgene plasmid 42229). The plant expression constructs and their target genes are provided in Table S1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: National Agri-Food Biotechnology Institute, Department of Biotechnology, Government of India, Mohali, Punjab, India 160071.

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.

Show MeSH