Neopyropia is an important economic macroalga and is one of the main macroalgae cultivation species in China, Japan, South Korea and other countries. As a critical juncture in the evolution of photosynthetic organisms, red algae to which Neopyropia belongs is regarded as a potential model species for studying the basic biology of macroalgae.

However, due to complex genome of extremely high GC content and uncharacterized regulation mechanisms of gene expression, the gene editing in Neopyropia, which is crucial for research of both genetic breeding and reverse genetics, is hard to realize.

Recently, the research team led by Prof. WANG Guangce from the Institute of Oceanology of the Chinese Academy of Sciences (IOCAS) made a significant progress in the reverse genetics research in economic macroalgae, and achieved gene editing in Neopyropia yezoensis, paving the way for molecular design breeding of economic macroalgae.

The study was published in New Phytologist on Sept. 12.

Researchers developed a gene editing strategy based on the life history of Neopyropia and optimized the CRISPR/Cas system. They introduced a self-cleaving peptide gene from porcine virus to link selection marker gene and CRISPR-associated protein 9 (Cas9) to enable high expression of Cas9 in hygromycin-tolerant strains.

Meanwhile, they identified and cloned the U6 gene and its promoter of Neopyropia for the first time, which can be recognized by RNA polymerase III to initiate the transcription of small nuclear RNA. Researchers used it for the expression of gRNA, putting the final piece in place for Neopyropia gene editing based on the CRISPR/Cas system.

The team also achieved precise knockout of targeted genes in Neopyropia and obtained a series of genome-edited strains with potential breeding application, the homozygous mutants accounted for 9.6%.

"The establishment of gene editing technology lays the foundation for reverse genetics research in Neopyropia," said Dr. XIE Xiujun, one of corresponding authors of the study. "It will not only promote Neopyropia yezoensis as a model species for basic biological research on macroalgae, but also provide an important tool for molecular design breeding of Neopyropia yezoensis."

Construction of DNA vectors for promotor verification and gene editing in Neopyropia yezoensis (Image by IOCAS)