Repositorium
Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA
208
Journal Article / 2015
Svitashev, Sergei; Young, Joshua K.; Schwartz, Christine; Gao, Huirong; Falco, S. Carl; Cigan, A. Mark
Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
476 |
Cigan, Mark A. USA |
Zea mays |
CRISPR/Cas9 MS26 |
Male sterility SDN1 |
Market-oriented Agronomic value |
477 |
Cigan, Mark A. USA |
Zea mays |
CRISPR/Cas9 MS45 |
Male sterility SDN1 |
Market-oriented Agronomic value |
478 |
Cigan, Mark A. USA |
Zea mays |
CRISPR/Cas9 ALS1 |
Herbicide tolerance SDN1 |
Market-oriented Herbicide tolerance |
479 |
Cigan, Mark A. USA |
Zea mays |
CRISPR/Cas9 ALS2 |
Herbicide tolerance SDN2 |
Market-oriented Herbicide tolerance |
480 |
Cigan, Mark A. USA |
Zea mays |
CRISPR/Cas9 LIG |
Disrupted formation of leaf cuticular wax (liguleless) SDN1 |
Basic research Basic research |
481 |
Cigan, Mark A. USA |
Zea mays |
Meganucleases LIG |
Disrupted formation of leaf cuticular wax (liguleless) SDN1 |
Basic research Basic research |