Targeted Genome Editing In Maize Using CRISPR-Cas9

Corn is one of the worlds most widely planted, within the scope of the highest yield of food crops, which makes important contributions to the world food security. In addition, it is also the important raw materials in the feed and industrial processes. Precise new methods to reveal the genetic mechanism of important agronomic traits for maize will be necessary if the corn to be fully harnessed to meet the burgeoning food, fiber, and fuel needs of an expanding world population under the pressure of decreasing agricultural resources and increasing of the harsh environment. Targeted genome editing, the ability to manipulate a genome precisely, site-specifically and permanently, has been an ideal way to maize genetic improvement. As the third generation of artificial site-specific nuclease, the RNA-guided CRISPR-Cas9 system is becoming a hot spot of the plant biology research, with much simpler design principles, facile construction process as well as the capability of multiplex gene editing. Here we configured the well-studied type Ⅱ CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) for targeted genome modification in maize, the main results were as follows:1. We configured the core components of the well-studied type Ⅱ CRISPR-Cas9 system from Streptococcus pyogenes. The open reading frame of Cas9 was maize codon-optimized and nuclear localization signal peptides were added. The expression and nuclear localization signal of Cas9 was confirmed in the maize protoplast. In addition, a functional maize U6 snRNA promoter was characterized to express the short non-coding targeting RNA, named as ZmU6-1.2. We established a database for CRISPR-Cas9 targets in exonic regions with minimally off-target loci matches. Targeted gene mutagenesis was detected for 76 loci by maize protoplast assay, with the cleavage efficiency ranged from 0.18% to 78.83%. In addition, the start purine nucleotide could be replaced by other nucleotides for the characterized ZmU6-1 promoter, which can be useful to increase the chance for ideal targets selections for specific genome modifications.3. We use ZmPSY1 genes as an example to study the mutation type and mutation rate induced by CRISPR-Cas9 in the stable express transgenic lines. And the heritability of the mutations was also studied. The mutation types induced by CRISPR-Cas9 were multiple, including deletions, insertions and deletions accompanied by insertions. The mutations produced by CRISPR-Cas9 system in the stable transgenic TO lines could be transmitted to the next generation, and CRISPR-Cas9 can be continuously active throughout the development of plant.4. In our study, in vitro cleavage analysis for all the six potential off-target sites located in gene regions for the RGN1 locus suggested that none of the putative off-target sites were modified. Furthermore, analysis at transcriptional level in our study suggested that constitutive expression of Cas9 brought no significant impact on the whole genome transcription. However, we also detected the possible off-target events with non-correspondence in genotype and phenotype, and related studies for confirmation is underway. Therefore, to establish a comprehensive evaluation system for CRISPR-Cas9 is necessary to facilitate its applications.