Genetics, Development and Cell Biology Ph.D. candidate — Si Nian Char

Major: Genetics

Major Professor — Bing Yang, GDCB affiliate faculty member (professor)

Title: Development and utilization of genome editing toolkit in maize, sorghum and rice

Abstract: Genome editing technologies have revolutionized the world of biology. Genome editing for site-specific genomic alterations started with engineered meganucleases, followed by programmable zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat associated proteins (CRISPR/Cas). The latter consists of CRISPR-Cas9, -Cpf1 (Cas12a), -C2c1 (Cas12b), -C2c2 (Cas13a) and CRISPR/Cas9 derived base editors (Adenine and Cytosine Base Editor), providing alternative toolkit for basic and applied research. Users have more options to edit, delete, insert, substitute, inactivate, activate, repress, label, methylate and acetylate the genetic elements (DNA or RNA) as they need.

In my presentation, I will focus on the development and utilization of TALENs, CRISPR/Cas9 and CRISPR/Cas12a targeted mutagenesis tools in plants. The proof of concept experiments using genome-editing tools in three major crops including rice, maize and sorghum will be presented. During the early days, we had successfully established the custom-tailored TALENs to target the endogenous gene, glossy2 in maize. The detailed methods from assembly of TALEN repeats to analysis and characterization of the inheritability and phenotype of glossy2-edited plants in T₁ generation will be described.

I will also showcase another more efficient Agrobacterium tumefaciens mediated CRISPR/Cas9 genome-editing method in maize. Our maize CRISPR utilizes A. tumefaciens to deliver the CRISPR/Cas9 constructs into maize immature embryos and target the Argonaute 18 and dihydroflavonol 4-reductase genes in maize. On this basis, I extrapolated this technology to other agronomically essential crops that feed the world such as rice and sorghum. Two quantitative trait loci (QTLs) for flowering time (SbFT) and plant height (SbGA2ox5) in sorghum had been tested by using CRISPR/Cas9. The editing efficiency can reach up to 70% in maize and 83.3% in sorghum depend on the targeted genes. Finally, the application of an optimized CRISPR/Cas12a technology in rice will be detailed in my presentation. Multiplexing CRISPR/LbCas12a constructs were Agrobacterium-delivered into rice mature embryo-derived calli to target eight lesion mimic candidate genes (Oslm 1 to Oslm 8). Taken together, genome editing technologies are robust and useful genetic tools to explore functional genomics in crop plants and for crop improvement.