CRISPR-Cas9-Mediated Genome Editing of the Model Grass Species Brachypodium distachyon

Abstract

The use of CRISPR-Cas9 to generate insertions or deletions (INDELS) resulting in stable functional knockout mutants of a targeted gene of interest is explored in the model grass species Brachypodium distachyon. Selection of a single-guide RNA based on location in the gene and low risk of potential off-target effects is initially presented, with the goal of generating INDELS in a single-gene target. Next, the use of the polycistronic t-RNA/gRNA system to simultaneously target multiple genes, or, alternatively, to generate a large insertion within a single gene, is discussed. Methods to transform chemically competent or electrocompetent Agrobacterium tumefaciens AGL-1 with CRISPR vectors, followed by stable transformation of Bd21–3 Brachypodium distachyon embryogenic callus, followed by plant regeneration, are outlined. Lastly, two methods for genotyping are described. The first is an inexpensive alternative based on PCR product fragment sizing analysis (within a single nucleotide difference to the wild-type sequence) to quickly ascertain the number of bases inserted or deleted on each allele of the targeted gene, which can then be followed by sequencing homozygous progeny of the initial regenerated transformants.