Application of CRISPR/Cas9 gene editing technology in fruit trees

Abstract: Since its development in 2013, the CRISPR/Cas9 has become a valuable genome editing tool that can cut DNA in specific regions of the genome, achieve specific changes in DNA and introduce new repair mechanisms, and precisely delete, replace or insert specific sequences at target locations in crop genomes. Compared with previous gene editing tools, this tool has obvious advantages such as simplicity, high efficiency, low cost and powerful function. At present, the application of CRISPR/Cas9 in citrus, grape, apple, banana, strawberry, kiwi, litchi, blueberry and other fruit crops has been reported.In this paper, various applications of CRISPR/Cas9 gene editing technology in fruit trees were described in detail, including albino phenotype, regulation of juvenile and flowering stage, regulation of fruit quality and creation of dwarf, disease-resistant and pest resistant varieties. And the problems and solutions of CRISPR/Cas9 gene editing technology were summarized. Compared with model plants, the application of CRISPR/Cas9 in fruit crops is limited due to more complex genome and less mature genetic transformation system. At present, CRISPR/Cas9 editing technology is mainly applied in fruit crops such as citrus, grape, apple, banana and strawberry, and genome editing of fruit trees such as kiwi,litchi and blueberry has also been reported. In fruit crops, related work has been carried out mainly around the following traits:(1) Albino phenotype, the phytoene desaturase(PDS) as an indicator gene of CRISPR/Cas9 editing technology, is a more direct target trait and has been knocked out in most fruit trees.(2) FTL and CEN genes were knocked out in apple and kiwifruit to regulate flowering stage, and early flowering mutant plants were obtained.(3) Breeding of varieties for resistance to pests and diseases through gene editing is the most widely used in fruit trees, especially in the study of resistance to citrus canker by knocking out CsLOB1 gene. In addition, banana resistant to wilt disease, grape resistant to Botrytis cinerea, cocoa resistant to phytophthora disease and litchi resistant to phytophthora have been developed.(4) CRISPR/Cas9 editing technique has also been used to create new plants with dwarfing phenotypes in bananas and pears.(5) Using gene editing technology to improve fruit quality, the regulation of the synthesis of fruit nutrients in bananas, apples, and grapes has been achieved, and the appearance and quality of the fruit have also been improved and optimized, such as producing whiteskinned strawberries, kiwis with reduced epidermal hair or without hair. In the process of genetic transformation of fruit trees, CRISPR/Cas9 vector almost infects plant tissues or organs through agrobacterium-mediated method. Besides agrobacterium-mediated protoplast transformation, PEG mediated protoplast transformation has also been applied in apple and litchi. There are some differences in the transformation materials used by different species. For example, suspended embryonic cells are usually used in bananas and grapes. Flower organs have also been reported to be used. The epicotyls is commonly used in citrus, and leaf disks are commonly used in strawberry and kiwi. Cas9 and sgRNA are the core parts of CRISPR/Cas9 editing vector. The expression of Cas9 and sgRNA is of decisive significance to the editing efficiency of vector. Most Cas9 promoters use the cauliflower mosaic virus promoter 35SCaMV and the corn ubiquitin promoter Pubi. Arabidopsis U3(AtU3) and U6 promoters(AtU6) and rice U3(OsU3) and U6 promoters(OsU6) are commonly used as promoters of sgRNA. Endogenous plant promoters U3 or U6 have been used in apple, citrus, grape and strawberry to activate and enhance the expression of sgRNA. It has been proved to be an effective means to improve editing efficiency. The efficiency of gene editing in fruit trees is often diverse among different fruit trees, and even there are great differences. Although CRISPR/Cas9 gene editing technology is a powerful and widely used tool, in fact, there are still some inevitable problems in the application process. First, it is difficult to establish conditions based on mature and stable tissue culture regeneration system and genetic transformation system. Second, miss target effect is a common problem. Three, more appropriate Cas9 and sgRNA promoters need to be selected. Therefore, continuous optimization of genetic transformation system and regeneration system is essential for the application of CRISPR/Cas9 in fruit trees. The selection of gene target sites and the design of sgRNA are very important in reducing miss efficiency. In addition, editing vectors usually design multiple sgRNAs to simultaneously target a gene to improve the accuracy of editing the target gene. Selecting suitable promoters to simultaneously enhance the expression of Cas9 and sgRNA is an important factor for high frequency mutation in fruit trees. In addition, emerging gene editing techniques could complement the CRISPR/Cas9 system, where single-base editing can precisely replace a single base, realizing C/G to A/T and C/G to A/T transformation, guided editing can realize all types of base editing and CRISPR/Cpf1 multi-gene editing system has more advantages than CRISPR/Cas9 system in multiple gene editing and chromosome fragment deletion.