A new fast and efficient strategy for building a pair of pairs of sgRNA Cas9 binary expression vectors.

Kiwi is an important global emerging fruit due to its rich nutritional value and unique flavor.
with the development of China and the world kiwi industry, how to quickly and efficiently create excellent characteristics of new species and cultivate new varieties, has become the key to restrict the development of industry.
, the CRISPR/Cas9 genome editing technology developed by clustered regularly interspaced-short-level-per-life-and-crisp-file-proteins, CRISPR/Cas9 genome editing technology has become an important tool for crop genetic modification and genetic function research, but there are significant differences in the efficiency of the system among different species.
in kiwi, there is currently no mature genome editing system available.
recently, Wang Zupeng, a doctoral student at the South China Botanical Garden of the Chinese Academy of Sciences, under the guidance of researcher Huang Hongwen and associate researcher Liu Yifei, established a new rapid and efficient construction strategy for the Cas9 dual expression vector of pairs of sgRNA, resulting in a sgRNA-into-sgRNA-cas9 vector containing four target sgRNA-based sg-s.
compared to the previous pair of sgRNA cloning methods, the strategy required only two sgRNA-containing primers to be synthesized, significantly reducing costs.
researchers further compared two different sgRNA expression box types -- the dosa-inverse tRNA-sgRNA system (PTG) and the traditional CRISPR expression box, for gene editing efficiency in kiwis.
results show edited that the target mutation efficiency of THE PTG/Cas9 system in kiwi is nearly 10 times higher than that of the traditional CRISPR/Cas9 system.
the study also found that both systems were able to successfully induce the albino phenotype of kiwi seedlings regenerated by G418 resistant injury tissue.
the first time that the study has established multiple and efficient PTG/Cas9 genome editing systems in kiwis, and the results provide a reference for optimizing genome editing efficiency in other plants or crops.
research was published in Plant Biotechnology Journal, a journal of plant biotechnology.
the research was supported by projects such as the National Natural Science Foundation of China and the Science and Technology Service Network Program (STS) of the Chinese Academy of Sciences.
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