CRISPR/Cas9-Based Genome Editing of Transcription Factor Genes in Marchantia polymorpha.

Plant transcription factors (TFs) belong to a wide variety of gene families. The systematic and rapid establishment of knockout lines of TF genes is critical for functional genetics. Genome engineering techniques for dissecting out the molecular function of TFs have been dramatically improved by CRISPR/Cas9-based genome editing technology. In the CRISPR/Cas9 system, Cas9 functions as a Cas9-gRNA ribonucleoprotein complex and uses its DNA endonuclease activity to induce the cleavage of the genome, which is targeted by gRNA. Double-strand breaks sometimes induce insertions and deletions at the target site, leading to frameshift mutations of TF genes. In this chapter, we describe a detailed protocol for the targeted mutagenesis of TFs using the CRISPR/Cas9 system, specifically in the case of Marchantia polymorpha, an emerging model plant for functional genomics. The CRISPR/Cas9 system is highly versatile for targeting genomic sequences because only an alteration of the gRNA sequence is needed to change target sequences. The labor and cost required to establish genome-edited lines are low enough that multiple mutants of TF genes can be generated in one laboratory. The CRISPR/Cas9-based genome editing technique consists of four steps: (1) gRNA design; (2) vector construction; (3) transformation; and (4) isolation of genome-edited lines. This manuscript focuses mainly on the strategy of gRNA design, the workflow for off-target searches, and the selection and identification of genome-edited lines by genotyping. Although we describe a protocol for M. polymorpha, the basic strategy of generating genome-edited lines of TF genes should be applicable widely to other plants.