A Highly Ecient Protocol To Establish Regeneration And Genetic Transformation Systems And Create Targeted Mutations Using CRISPR/Cas9 in Lycium Ruthenicum

Background: The CRISPR/Cas9 system has rapidly become the preferred tool for various biological sequencing projects due to its high eciency, specicity, simplicity and versatility, and it has been utilized for targeted genomic alternations in several important plants of Solanaceae, including tomato, tobacco, potato, petunia and groundcherry. Wolfberry is the sixth most important solanaceous crop in China following potato, tomato, eggplant, pepper and tobacco. To date, there has been no report on CRISPR/Cas9 technology to improve Lycium ruthenicum due to the unknown genome and the lack of ecient regeneration and genetic transformation systems. Results: In this study, we established a suitable regeneration and genetic transformation system of Lycium ruthenicum, the fw2.2 gene was identied, which was the rst fruit weight gene identied from tomato and accounted for 30% of the variation in fruit size. The gene editing of black wolfberry were carried out by CRISPR/ Cas9 for the rst time here with a very high editing eciency (95.45%) in fw2.2. Four homozygous mutations and nine biallelic mutations were obtained from T0 generation plants. Conclusions: These results suggest that the CRISPR/Cas9 system is effective for gene editing study of black wolfberry, and we expect this approach to be routinely applied to this important economic fruit.


Background
Wolfberry is used as a traditional medicine in China and other Asian countries owing to the abundance of anthocyanins, trace minerals, vitamins and polysaccharides in the fruit [1,2]. There are about 160 000 hm 2 of production area and 410 000 tonnes of yield with 18.7 billion RMB value in China in 2018. It was thought Black Chinese wolfberry has more important value than other fructus lycii with procyanidine-rich content, as a result, the arti cial cultivation of Lycium ruthenicum has been greatly developed in North West China. The cultivation of black wolfberry is also labor-intensive industry and the fruit is mostly hand harvested. Small fruit and dense sharp thorn make it more di cult for harvest. The large fruit size is an important fruit quality characteristic which affects its market competitiveness and nutritional value. As a producer, obtaining new large-fruited cultivars is a major breeding goal for grower pro tability. Therefore, understanding the genetic background of fruit size-related genes will signi cantly improve the breeding e ciency of large-fruit varieties, which will also greatly promote the use of small-fruit wild germplasm, as it will reduce the number of generations required to obtain the commercial fruit size needed for a new cultivar. The regulation of fruit size by genetic changes is the most widely studied in tomato to date (Solanum lycopersicum L.) [3,4]. fw2.2 was the rst fruit weight gene identi ed from tomato as a result of domestication, it is a major quantitative trait locus that regulates fruit size and weight in tomato, and natural genetic variation at this locus alone can change the size of fruit by up to 30% in tomato between large, domesticated tomatoes (Lycopersicon esculentum Mill.) and their small-fruited wild relatives [5].The "small-fruit" alleles at the fw2.2 locus exists in all wild tomatoes, whereas all cultivated tomatoes tested are xed for "large-fruit" alleles [6]. The higher fw2.2 transcript levels negatively regulate cell division, which result in a decrease in cell number, therefore affect the mitotic activity during early fruit development [5,7]. Many studies have demonstrate that cell number is a key factor determining fruit size in fruit crops including peach [8], sweet cherry [9], olive [10], and apple [11]. Due to the critical roles of fw2.2 in regulating cell number, orthologs to these fw2.2 genes for fruit sizes are further studied [12,13]. Therefore, fw2.2 was selected for establishing the technology of gene editing in Lycium here.
The CRISPR/Cas9 system has been utilized for targeted genomic alternations in several important plants of Solanaceae, including tobacco, tomato, potato, petunia and groundcherry [14,15]. Among horticultural crops, tomato has received much more attention regarding genome editing than other crops: ~42% of genome-editing studies have involved tomato, whereas ~ 13% have involved potato [15]. CRISPR/Cas9mediated knock-out of polyphenol oxidase genes in eggplant has also been reported [16]. However, this powerful tool for genome editing has not yet been used in Lycium.
In this study, we established an e cient regeneration and genetic transformation system for Lycium ruthenicum, and we rst adapted a schematic work ow and cloning strategy for black wolfberry CRISPR/Cas9 system in order to breeding elite cultivars with bigger fruits in facing on the harvest di culty since the stems are full of thorn. The results show that this CRISPR/Cas9 system is effective in Lycium ruthenicum.

Plant materials
Wild Lycium ruthenicum seeds were obtained from Alxa, Inner Mongolia, and rinsed under running water for 2 hours, then 75% alcohol was used to disinfect for 30s, rinsed with sterile water once, then rinsed with 4% sodium hypochlorite for 3 minutes, the seeds were rinsed with sterile water 3 times and then inoculated in 1/2MS medium, placed at 25°C and 14h/d light (1500~2000lx) in the tissue culture room to obtain tissue culture seedlings for later gene editing study. were used as seed sequences to search the Lycium ruthenicum transcriptome sequencing database (SRA:SSR7700825), and a highly homologous sequence was obtained based on BLAST search. The Pfam database (http://pfam.janelia.org/) [30] and the Conserved Domain Database (CDD) of the NCBI [31] were used to analyze the obtained sequence that met the known conserved domains and motifs requirements. The RNA and genomic DNA were extracted from Lycium ruthenicum using Tiangen Kit, and the gene-speci c primers were designed to amplify the fw2.2 sequence (Additional le 1 Table S4). The exons and intron regions were analyzed after the full length of the fw2.2 gene was got. The sequences of fw2.2 proteins producing signi cant alignments were searched on BLASTP programs (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) to construct phylogenetic tree, and the neighbor-joining (NJ) method of the MEGA-X was used to analyze phylogenetic relationships, the con dence limits of each branch in the phylogenetic tree were assessed by 1000 bootstrap replications and expressed as percentage values.

Optimization of regeneration system and Agrobacterium-mediated transformation of Lycium ruthenicum
The leaves of the Lycium ruthenicum tissue culture seedlings were cut into about 0.5 cm and then transferred to MS medium (A1-A12) supplemented with various concentrations and combinations of 6-BA, NAA and 2, 4-D to induce callus (Additional le 1 Table S1). The callus induction rate was counted after 30 days based on three experiments using 30-40 explants in each treatment. The callus was subsequently transferred to MS medium (B1-B10) supplemented with various concentrations of 6-BA and NAA to induce differentiation (Additional le 1 Table S1). 30-40 explants were used in each treatment of three experiments, and explants were subcultured at intervals of 20 days. The differentiation rate and multiplication coe cient were counted and recorded after 60 days. The optimal regeneration system of Lycium ruthenicum was determined based on the above callus induction and differentiation experiments.
The leaves of Lycium ruthenicum tissue culture seedlings were used for Agrobacterium transformation. The in uence of hygromycin concentration, infection time, co-cultivation time, bacterial solution concentration and acetosyringone concentration were comprehensively analyzed to explore the optimal genetic transformation conditions (Additional le 1 Table S3) . The Agrobacterium cells were collected and resuspended in MS liquid medium after expanded cultivation in LB medium containing 50mg/L Kan, 50mg/L Gen and 50mg/L Rif. After infecting in a sterile test tube containing Agrobacterium suspension, the leaves of Lycium ruthenicum were transferred to co-culture medium (MS+0.5mg/L6-BA+0.5mg/LNAA) for two days in the dark at 25℃, and then transferred to resistant callus selection medium (MS+ 0.5mg/L6-BA+0.5mg/LNAA+40mg/L Hyg+200 mg/L Carb). After that, the resistant callus were transferred to the medium (MS+0.2mg/L6-BA+0.05mg/LNAA+40mg/L Hyg+200 mg/L Carb) to obtain resistant buds. With 2 rounds of selection and culture, the resistant buds were cut and transferred to rooting medium (1/2 MS+200 mg/L Carb) to obtain the gene editing tissue culture seedlings of Lycium ruthenicum.

Identi cation of gene-edited Lycium ruthenicum
The unedited tissue culture seedling was used as a control, the genomic DNA was extracted as a template for ampli cation and sequencing. The detection primers were designed in the upstream and downstream of the gene editing site (Additional le 1 Table S7). The mutation type can be observed from the sequencing chromatograms ( Fig. 6 and Additional le 2), when the target sequence has a heterozygous mutation (only one chromosome is mutated, while the other is not) or biallelic mutations (different mutations on two chromosomes), there will be overlapping peaks after the target site. When the target sequence has a homozygous mutation (two chromosomes have the same mutation) or no mutation, a single peak appears. The mutation sequences can be read directly from the sequencing les. Based on this, the mutation types were counted, and the editing e ciency were also counted following the formula: gene editing e ciency (%) = (number of gene-edited seedlings / number of resistant seedlings ) × 100%.

Results
Analysis of fw2.2 gene structure and phylogenetic analysis of Lycium ruthenicum The full length of the DNA of Lycium ruthenicum fw2.2 is 2323bp, it is divided into three exon regions by introns, the lengths of which are exon 1 :259bp, exon 2:210bp, exon 3 :80bp. The cDNA sequence is 549bp in length, with a total coding of 182 amino acids. The fw2.2 gene of Lycium ruthenicum has a PLAC8 (Placenta-speci c 8) conserved domain ( Fig. 1a), and belongs to the same family as fw2.2 of other plants. After BLASTp alignment on NCBI, some homologous protein sequences are obtained. Most of them were FWL/CNR family proteins, and few of them are PCR (Plant cadmium resistance) family proteins ( Fig. 1b). The two family proteins have the same conserved domain PLAC8 and similar structures. The selected Solanaceae fw2.2 homologous protein including Lycium ruthenicum fw2.2, Lycopersicon fw2.2, Physalis POS2, and Capsicum CNR1 are clustered in the same branch, and the amino acid sequence similarity is more than 85% ( Fig. 1C), indicating that fw2.2 protein is relatively conserved in Solanaceae. FW2.2-like genes have been renamed as the Cell Number Regulator (CNR) family, and it has a negative regulatory effect on cell number [17,18]. Studies have shown that fw2.2 can explain 30% and 47% of the fruit size phenotypic variation in Lycopersicon pimpinellifolium and Lycopersicon pennellii, respectively [19]. The POS2 (physalis organ size 2) in Physalis oridana encodes a putative ortholog of fw2.2, which can regulate the cell cycle and has a negative effect on fruit size [20]. The function of CNR1 in pepper is unknown. Tomato fw2.2 and Physalis POS2 both have the functions of regulating cell division and affecting fruit size. It is speculated that Lycium ruthenicum fw2.2 which has a close phylogenetic relationship with them may also have similar functions.

Establishment of Lycium ruthenicum regeneration system
The seeds of Lycium ruthenicum are disinfected and inoculated on 1/2 MS medium, we have established a suitable regeneration system of Lycium ruthenicum after the induction and differentiation of callus, and the rooting of regenerated seedlings (Additional le 1 Table S1). Although the callus induction rate in each medium is 100% after 15 days of callus induction, the callus growth status is different. Among them, callus grows best on A3 medium (MS + 0.5 mg/L 6-BA + 0.5 mg/L NAA) with green appearance, loose structure, no browning, and low vitri cation, which is the most suitable medium (Additional le 1 Table S1). The callus was subsequently transferred to the medium B1-B10 to induce differentiation, and the differentiation rate and multiplication coe cient were counted after 30 days. We found that the callus did not differentiate or the differentiation rate was low when it was cultured on the medium without 6-BA (B5) or the concentrations of 6-BA were higher (B1-B4 ). Both the differentiation rate and the multiplication coe cient were signi cantly increased on the medium supplemented with low concentration of 6-BA (less than 0.5mg/L). When the callus was cultured on B7 (MS + 0.2 mg/L 6-BA + 0.05 mg/L NAA) medium for cluster shoot induction, the differentiation rate and multiplication coe cient were the highest, which were (95.83 ± 2.23 )% and (7.06 ± 0.22)% respectively. The callus did not brown and the degree of vitri cation was relatively low (Additional le 1 Table S1). The differentiated shoots were then inoculated on 1/2 MS medium without hormones, and rooting can be induced after 15 days.

Target site selection and sgRNA Design
The CRISPR online design tool (http://crispr.dbcls.jp/ ) was used to determine the location of the target, the genome of tomato ( Solanum lycopersicum genome, SL2.40 ), a species with high homology with Lycium ruthenicum, was used as a reference to improve target speci city. Based on the selection criteria that the PAM site sequence is NGG, the GC content is 40%-70%, the target sequence avoids spanning intron regions and the occurrence of more than 4 consecutive T bases, the top sgRNA were selected for the knockout experiments. The sgRNA used in this study are shown in Table S2. The position of fw2.2-1 and fw2.2-2 is 1857-1879 and 171-193 respectively, spanning 1664 bases.
Establishment of Lycium ruthenicum genetic transformation system and e ciency evaluation of CRISPR/Cas Two single sgRNAs and a dual sgRNAs (sgRNA1 and sgRNA2) of fw2.2 were designed that target different sites after the whole sequence being cloned and phylogenetic analyzed. The target site of fw2.2-sgRNA1 and fw2.2-sgRNA2 is located in exon 2 and exon 1 respectively ( Fig. 2a). The high e cient Agrobacterium tumefaciens-mediated transformation of black wolfberry was performed here using the leaves under the condition of 0.2 Agrobacterium concentration (OD600), 10 min infection time, 200 μmol/L acetosyringone concentration and 2d co-culture time (Additional le 1 Table S3). The 40 mg/L hygromycin selection marker plus 200 mg/L carbenicillin were used to inhibit Agrobacterium growth. The entire experimental cycle took approximately 2 months from incubation to mutant identi cation with 2d co-cultivation, 15d calli production, 30d differentiation and sub-culture (Fig. 2b). The details of the transformation media can be found in Fig. 2c. The mean transformation e ciencies of these three lines were 2.66%, 1.18% and 5.33%, respectively.
In this study, a lot of resistant seedlings were produced. Twenty-one out of twenty-two, six out of eleven and fteen out of sixteen bigger plants were detected with novel mutations in the sgRNA1, sgRNA2 and sgRNA1/sgRNA2 regions. As shown in Fig. 2d, the gene editing e ciency of fw2.2-1 target is high (95.45%), while that of fw2.2-2 target is low (54.55%). However, the editing e ciency of homozygous mutants (18.18%) and biallelic mutants (9.09%) is higher than those of fw2.2-1 (4.55% ).
The dual-sgRNA CRISPR/Cas9 system was highly reproducible and highly e cient since it could result in more reliable loss-of-function alleles that lack a large essential part of the gene [21] . Here we found the editing e ciency of fw2.2 in the homozygote/biallelic mutations altogether (56%) by the dual-sgRNA CRISPR/Cas9 system is more than twice (27%) of that by the sgRNA CRISPR/Cas9 system though the editing e ciency of the dual-sgRNA system is only 93.75%,which is a little less than that of (95.45%) the sgRNA1 system (Fig. 2e&f). It was also found that there was a 1281 bp segment deleted and 29bp insertion in the fw2.2 of a T0 plant (Fig. 2g), which is similar to the result such as 934-bp deletion mutation at the AtMIR169a locus of Arabidopsis [21] .
Expression analysis of fw2.2 in gene-edited seedlings by quantitative real-time PCR We found that the expression of the fw2.2 gene changed signi cantly (Fig. 3). Among the selected geneedited seedlings, the expression of fw2.2 gene decreased most signi cantly in X21, which had a large deletion of 1281 bp, and its expression was only 0.01. X9 is a homozygous mutant, the expression of fw2.2 gene in which was only 0.03, and followed by X17 and X20, which were 0.06 and 0.15 respectively. When a gene expression cassette is introduced into a genome by CRISPR/Cas9, the sgRNA target gene becomes inactive because of disruption of the gene, which probably in uences the gene expression. These data suggest that CRISPR/Cas9 applied to fw2.2 had a signi cant effect on reducing gene expression , which probably in uences the developmental characteristics of the modi ed strain if the inactive gene is generally involved in metabolism.

Discussion
Until now, successful genome editing mediated by CRISPR/Cas9 was demonstrated in Solanaceae, including tomato [22], potato [23], tobacco [24], Petunia [25], Physalis [14] and eggplant [26], fully demonstrating the broad prospects of molecular genetic technology for crop improvement. However, due to the complex genomes, slow growth cycles, di culties of transformation and a lack of genomic information, it remains a long and arduous task to apply CRISPR/Cas9 to Lycium ruthenicum. In this study, we established a suitable regeneration and genetic transformation system for Lycium ruthenicum, which laid the foundation for the subsequent successful transformation of CRISPR/Cas9 plasmids.
Studies have shown that simultaneous targeting of multiple regions of the same gene or functionallyredundant genes can improve gene editing e ciency [27]. We constructed double target editing vector pCAMBIA1300-fw2.2-1/2 except for the single-target editing vectors pCAMBIA1300-fw2.2-1 and pCAMBIA1300-fw2.2-2. And both target sites were selected by the CRISPR online design tool using the tomato genome as a reference to improve speci city. Different mutation including homozygous, biallelic and heterozygous were obtained, and it was found that the mutation type with the highest proportion was 3 base deletion occurring at 3-5bp upstream of PAM site (Fig.2h).The deletion of the 1281 bp large fragment and 29bp insertion in the fw2.2 (Fig. 2g) were also found in the T0 plant. All these results suggest that the CRISPR/Cas9 system applied to Lycium ruthenicum is effective.
A recent report has demonstrated fw2.2 which encodes a negative cell number regulator increases fruit weight by approximately 30% between the domesticated tomato and its wild relatives in the genus [5]. The overexpression of ZmCNR1 and ZmCNR2 (members of the fw2.2 gene family in corn ) resulted in the decrease in plant and organ size [28]. The developmental expression pro le of Pafw2.2-like, a fw2.2/CNR family member in avocado revealed that the transcript levels of Pafw2.2-like were markedly higher in small fruit tissues than in normal fruit tissues [12]. Scorza et al. [8] found that the large-fruited peach exhibited a higher number of cells at all developmental stages comparing with small-fruited peach. Olmstead et al. [9]reported that the increases in cell number rather than cell size resulted in the difference of cherry fruits in the process of domestication and modern breeding. Franceschi et al. [13] identi ed the homologous genes PpCNR12 and PpCNR20 of fw2.2 in cherry and speculated that they were involved in fruit size regulation. More and more studies have supported that fw2.2 plays an important role in fruit size. In our study, the fw2.2 gene of Lycium ruthenicum that we identi ed has high homology with other species, studies have shown that speci c fw2.2-like proteins might share a similar biological function with the tomato fw2.2[28, 29], which suggested that fw2.2 in Lycium ruthenicum might be associated to regulation and/or repression of fruit cell division. Whether we could produce the bigger fruit phenotypes of black wolfberry or not needs to wait until next summer.

Conclusion
This study is the rst to use CRISPR/Cas9 technology for targeted genome editing in Lycium ruthenicum, and demonstrates that the system has high editing e ciency. The results obtained in this study can help to understand the mechanism of fruit development. Meanwhile, the regeneration and genetic transformation system established in this study can provide theoretical guidance for transgenic research of Lycium ruthenicum. The targeted knockout of desired genes using CRISPR/Cas9 is also of great signi cance for developing new traits of Lycium ruthenicum. The effect of successful editing on fruit development needs further morphological analysis and transcriptome sequencing.

Declarations
Not applicable.

Consent for publication
Not applicable.

Con ict of interest
The authors declare no con ict of interest.  Genome editing in Lycium ruthenicum using CRISPR/Cas9 technology. a. fw2.2 gene structure and sequences of the target sites. Black boxes: exons; grey lines: introns; sgRNA target sites and the PAM regions (Red). b. The process of transformation (Two-week-old callus; differential shoots after four-week of subculture; elongated shoots after six-week of subculture; eight-week-old rooted transformant). c. The media information used in the transformation. d. The gene editing rate of different fw2.2 target sites in Lycium ruthenicum. e. The editing e ciency of the dual-sgRNA system. f. Comparison of editing rate between constructs with different number of sgRNA cassettes. g. The combination of large fragment deletions with insertions in a T0 plant. The 1515 bp in italic is the intron. 1281bp deletions are labeled in gray. 29bp insertions shown in yellow are almost the inversion of the fragment nearby labeling with green. h. Editing type and preference of different target sites.   Dual target vector construction