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Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

Bibliography:

Haroon Butt, Ayman Eid, Zahir Ali, Mohamed A. M. Atia, Morad M. Mokhtar, Norhan Hassan, Ciaran M. Lee, Gang Bao and Magdy M. Mahfouz. Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2017.01441

Authors:

Haroon Butt, Ayman Eid, Zahir Ali, Mohamed A. M. Atia, Morad M. Mokhtar, Norhan Hassan, Ciaran M. Lee, Gang Bao and Magdy M. Mahfouz

Keywords:

Genome engineering, CRISPR/Cas9, HDR, RNA-templated repair, gene editing

Year:

2017

Abstract:

‚ÄčThe CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

ISSN:

1663-4365