CRISPR-Cas9 emerged as a powerful tool for gene editing, which has been widely used in plant functional genomics research and crop genetic breeding (Chen et al. 2019). The target specificity of CRISPR-Cas9 relies on the 20-base-pair single guide RNA (sgRNA), making it relatively quick and straightforward to create plant-specific mutant libraries through large-scale synthesis of sgRNAs targeting multiple genes or even the whole genome. Several CRISPR-Cas9 mutant libraries have been developed for crops such as rice (Lu et al. 2017; Meng et al. 2017), soya bean (Bai et al. 2020), Brassica napus (He et al. 2023), and cotton (Sun et al. 2023). However, no CRISPR-Cas9 mutant library has yet been generated in woody crop plants. Grape (Vitis vinifera L.) is one of the oldest and most economically valuable fruit crops worldwide. The MYB family is one of the most abundant and versatile transcription factor families in plant (Wu et al. 2022). Here, we described a strategy for generating a collection of MYB mutant lines in grape using a sgRNA library.

We obtained 138 grape MYB transcription factor sequences from the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/family.php?sp=Vvi&fam=MYB) (Appendix A). Since genes with similar sequences often share similar functions, a phylogenetic tree was constructed and divided all MYBs into 30 sets based on the sequence similarity (Fig. 1-A and Appendix B-a). A total of 127 sgRNAs were designed, each targeting conserved regions shared by two or more MYB transcription factors with fewer than three base-pair differences. This approach aimed to simultaneously mutate multiple MYB members within a cluster using a single sgRNA, addressing the challenge of genetic redundancy (Appendix B-b). These shared target sites were designated as Target1 (T1). In addition, specific sgRNAs targeting individual MYB transcription factors were designed using the  CRISPR-P 2.0 online tool (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR) (Liu et al. 2017). The design criteria included selecting target sites within exons near the start of open reading frames (ORFs), a GC content of at least 40%, and an off-target efficiency below 0.4. Finally, 138 sgRNAs targeting specific sites for each MYB transcription factor were designed, referred to as Target2 (T2). In total, a comprehensive sgRNA library comprising 265 sgRNA was developed to target 138 MYBs, with an average  of 1.92 sgRNA per MYB (Fig. 1-A and Appendix C).

The sgRNA fragments from the same set were mixed as one sgRNA pool, ligated into the pKSE401 vector using Gibson ligation, and subsequently transformed into the Escherichia coli  TOP10 competence cells (Fig. 1-B and Appendix D-a). To evaluate the ligation efficiency of the sgRNA pool with the vector, 90 E. coli clones from sets #1-3 were randomly selected and sequenced. The results demonstrated that the ligation efficiency exceeded 90% and the sgRNA coverage ratio over 80%, confirming the feasibility of this method (Appendix D-b). Using this approach, approximately 1300 (~5×) positive E. coli clones were obtained across the 30 sets (Fig. 1-D). Plasmids extracted from each set were mixed in equal proportions and transformed into Agrobacterium tumefaciens GV3101 competence cells. Finally, all Agrobacterium colonies were collected and verified with next-generation sequencing (NGS). The results revealed that 95.31% of the sequences in the library were accurate, and 178 of 265 sgRNAs were represented by at least one read, targeting 125 (90.58%) MYB transcription factors. Most (83.93%) sgRNA read counts fell within the range of 2⁸-2¹⁵. These results indicated that the sgRNAs library in Agrobacterium exhibited high accuracy and gene coverage, which is usable for grape transformation (Fig.1-E).

Vitis vinifera L. cv. Cabernet Sauvignon is one of the most renowned red wine grape varieties, widely cultivated worldwide. In this study, pro-embryonic masses of ‘Cabernet Sauvignon’ were used as recipient material for Agrobacterium-mediated transformation (Fig. 1-C and Appendix E). A total of 1354 kanamycin-resistant seedlings were obtained, and which 341 were confirmed as transgenic lines (PCR positive). And, all the lines were determined to harbor a single correct sgRNA, representing 13 unique sgRNAs targeting 18 MYB transcription factors. Target site DNA was amplified and sequenced, revealing only 67 gene-edited lines with mutations in 8 MYB transcription factors (Fig. 1F and Appendix F and G). Among these, 56 lines were chimeric mutants, nine were biallelic mutants, one was a homozygous mutant and one was a heterozygous mutant (Appendix H). Five Target1 type transgenic lines were obtained, three of them did not mutate in all of the targeted genes, in which the sgRNA was targeting two or more completely conserved sites. Additionally, gene-edited lines for GSVIVT01032467001-T1 and GSVIVT01014770001-T1 were producted. However, the sgRNA harbored in these transgenic lines only caused the mutations in GSVIVT01032467001 and GSVIVT01014770001, without off-target effects on genes with similar sequences (Appendix I and J). All MYB-edited lines were subsequently transplanted into a greenhouse for observation (Appendix K). Phenotypic analysisrevealed that the GSVIVT01026481001 edited lines exhibited significantly enhanced tolerance to drought stress (Fig. 1-G).

CRISPR-Cas9 has greatly accelerated gene function research and breeding in plants. In this study, we developed a strategy for generating a collection of MYB mutant lines in grape using a CRISPR-Cas9 library. However, the relatively lower transformation efficiency in grape limited the number of mutants obtained. Factors affecting grape transformation efficiency primarily included the regeneration rate of the recipient material and the efficiency of Agrobacterium infection. Numerous studies have demonstrated that plant regeneration efficiency enhanced using developmental regulators such as BABY BOOM (BBM), WUSCHEL (WUS), GROWTH-REGULATING FACTOR (GRF), and REGENERATION FACTOR (REF) (Debernardi et al. 2020; Yang et al. 2022). Additionally, plant transformation and gene editing efficiency improved through optimized genetic transformation methods and gene editing vector designs (Debernardi et al. 2024; Yan et al. 2024). We confirmed that a large number of edited plants could be obtained simultaneously using a sgRNA mixed-pool library, provided that grape transformation efficiency is improved. This strategy holds significant potential for constructing genome-wide mutant libraries in woody crop plants in the future.