Production of homozygous deletion mutants targeting fertilization regulator genes through multiplex genome editing

Abstract

Producing the gene-knockout mutant is a critical strategy in reverse genetics for gene functional analyses. Plant science has applied various mutagenesis, including chemical mutagen, T-DNA insertion, and genome editing. The intact gene expression is completely disrupted, while the mutant retains the intact sequence region. Lately, effective whole open reading frame (ORF) deletion through the CRISPR/Cas9 system using multiplex guide RNAs was reported in Arabidopsis, targeting a gene expressed in somatic cells. Here we applied the scheme targeting the reproductive genes GENERATIVE CELL SPECIFIC 1(GCS1), GAMETE EXPRESSED 2 (GEX2), DUF679 DOMAIN MEMBRANE PROTEIN 8 (DMP8), and DMP9, which are fertilization regulators. Homozygous gene deletion lines were successfully obtained in the T1 generation, with acquisition rates ranging from 8.3% to 30.0%. The rates approximately correlated with the scores predicted by the DeepSpCas9. The analysis of the GCS1 deletion line ({Delta}gcs1) suggested that avoiding the first bolt removal is important for consistent phenotype of the developed inflorescences. Compared to the previously reported mutants, the GEX2 deletion line ({Delta}gex2) showed a difference in seed development phenotype, indicating that the remaining intact gene region in the mutant could unexpectedly influence the function. Simultaneous deletions of DMP8 and DMP9, which are in distinct chromosomes, were also succeeded in the T1 generation. The obtained results showed that all deletions were inheritable. The scheme challenged here demonstrated the effective production of homozygous mutants for the genes, even if the reproductive lethal or recessive.