TALENs and CRISPR/Cas9 System for Targeted Genome Modification in Maize

After the maize genome sequencing was completed in 2009, maize functional genomics became an attractive research area. While it is relatively easy to obtain gene-specific mutants in plants with simpler genomes, the mutant creation process is much more difficult and time consuming in maize. As the most straightforward way of studying a gene is to create a mutant, one possible approach is through targeted mutagenesis. Two widely used systems, the TALEN (transcription Activator Like Effector Nuclease) and the CRISPR/Cas9, have been successfully used in a few plant species. Here, we apply these two technologies to carry out targeted genome modification and functional analyses of selected genes in maize. In total, we selected 8 targeting sites in 5 genes for TALEN-expressing constructs and 6 targeting sites in 3 genes for gRNA-Cas9 expressing constructs. We first tested the binary vector products in maize protoplasts and then transformed the vectors via agrobacterium or particle bombardment using immature embryos. We were able to detect nucleotide addition or deletion mutants in a number of target sites in both protoplast and callus. The average mutation ratio in the callus is around 10% for CRISPR/Cas9 system. Further work is on-going to develop robust, reliable protocols for maize mutagenesis.