Using Two Guide RNAs to Study Gene Functions in Arabidopsis thaliana

Efficient generation of heritable mutations in Arabidopsis was achieved using CRISPR/Cas9, producing bi-allelic mutations in the T2 generation.

• The CRISPR/Cas9 technique enables the creation of knock-out alleles for any gene and entire gene families in plants.
• Bi-allelic mutations were observed primarily through the canonical non-homologous end joining pathway.
• Inherited mutations resulted from a different repair pathway linked to polymerase θ, known as synthesis-dependent microhomology-mediated end joining.
• A dual sgRNA approach was utilized, enhancing the reliability of generating loss-of-function alleles by targeting a gene with two sgRNAs.
• The streamlined CRISPR/Cas9 workflow could facilitate the development of true null alleles across the Arabidopsis genome.

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