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POSTERS: Biotechnology and genetic engineering

Genome editing of citrus using the CRISPR-Ribonucleoprotein complexes
Ali Parsaeimehr - Citrus Research and Education Center, University of Florida. Nian Wang- Citrus Research and Education Center, University of Florida

Genetic improvement of citrus using conventional breeding is a lengthy and challenging process due to the complex reproductive biology of citrus, including sexual incompatibility, highly heterozygous nature, nucellar seedlings, male or female sterility and a long juvenile phase. Biotechnology approaches such as CRISPR-mediated genome editing have the potential to accelerate the citrus improvement process. Heritable citrus genome modifications have previously been conducted via transgenically expressing Cas9/sgRNA in planta. Genetically modified plants generated via the transgenic expression of Cas9/sgRNA contain foreign DNA sequences, requiring a rigorous deregulation process before commercialization. Non-transgenic genome editing of plant can be conducted by transient expression of the CRISPR-Ribonucleoprotein (RNP) complexes in embryogenic plant cells.Here, we expressed, isolated and directly delivered three types of Cpf1 proteins from Francisella novicida (Fncpf1), Lachnospiraceae bacterium (Lbcpf1), and Acidaminococcus spp (Ascpf1) with the corresponding CRISPR RNAs (crRNA) using polyethylene glycol-mediated transformation and biolistic delivery methods. crRNAs targeting the canker susceptibility gene CsLOB1were used. Our results showed that Fncpf1 had the highest activity and is compatible and active with the Ascpf1 and Lbcpf1 crRNAs. Our results indicate that CRISPR?Cpf1 RNPs direct delivery is a potent method to generate foreign DNA-free genome edited citrus.