Detection of Target Sites Edited in Upland Cotton By The CRISPR/Cas9 System Mediated By Agrobacterium Rhizogenes


 Background

CRIPSR/Cas9 gene editing has the ability to effectively modify plant genomes. Multiple target sites usually were designed and the effective target sites were selected for editing. However, upland cotton is allotetraploid and is commonly considered as difficult and inefficient to transform. Therefore, it’s important to quickly identify feasibility of the target site. In this study, we use Agrobacterium rhizogenes K599 strain to infect cotton shoot meristem and induce them to grow hairy roots to detect the feasibility of a selected target designed in GhMYB25-like gene.
Results

We designed a sgRNA within the second exons of GhMYB25-likeA and GhMYB25-likeD and constructed the CRISPR vector. Transient hairy root transformation using A. rhizogenes K599 with four OD600s (0.4, 0.6,0.8, 1.0) was performed in Coker 312 (R15). The results show that A. rhizogenes at OD600 = 0.6–0.8 is the best concentration range for inducing cotton hairy roots. The other three cultivars (TM-1, Lumian 21, Zhongmian 49) were injected using A. rhizogenes K599 with OD600 = 0.6-0.8 and all produced hairy roots. We characterized ten R15 plants with hairy roots and detected different degrees of base deletions and insert at the target site in five R15 plants.
Conclusion

Overall, our data show A. rhizogenes-mediated transient hairy root transformation offers a rapid and efficient method to detect sgRNA feasibility in cotton.


Introduction
The CRISPR/Cas9 system is the latest generation of genome editing technology for site-speci c editing to study gene function and improving crop traits [1]. Its working principle is that the nuclease Cas9 protein cuts the target gene under the guidance of the guide RNA, resulting in double-strand breaks. During the repair of the double-strand DNA, the insertion and deletion of bases will be directed to achieve the sitedirected mutation of the target gene [2]. Compared with other genome-editing systems, CRISPR/Cas9 system has obvious advantages such as simple construction, low cost and easy implementation in laboratory, so it has been widely used in genome editing of Oryza sativa [3], Triticum aestivum [4], Solanum lycopersicum [5] and many other plants.
Agrobacterium rhizogenes (Rhizobiaceae), a gram-negative soil bacterium, can naturally infect most eudicots, a few monocotyledons and some gymnosperms and can induce the production of hairlike roots in plant wounds [6]. These so-called hairy roots have the advantages of rapid growth, multiple branches, and improved, stable production of secondary metabolites, so the transformation system has been widely used to produce important secondary metabolites [7,8], verify the function of genes related to root development [9], and study plant-microbe interactions [10]. Compared with Agrobacterium tumefaciens, A. rhizogenes has a wider host range, so there is no speci c selection of host or genotype. More importantly, the time between infection by A. rhizogenes and subsequent differentiation of roots is shorter than with A. tumefaciens, and thus better suited for rapid veri cation of genetic transformation. Studies have shown that A. rhizogenes can induce adventitious roots in a variety of plants such as Solanaceae [11] and Leguminosae [12].
Cotton is an important source of ber, protein and edible oil, and has great economic value. However, the complex genome characteristics of allotetraploid cotton poses a challenge to the analysis of cotton gene function and to transgenic breeding [13]. Due to the long genetic transformation cycle for cotton, complicated genetic transformation process and low transformation e ciency, the CRISPR/Cas system itself has off-target effects. Therefore, evaluating the feasibility of the selected target site is very important for successful genome editing of cotton. In this study, GhMYB25-like gene was selected as the target gene to construct CRISPR/Cas9 vector, and cotton shoot meristem produced hairy roots after infected by A. rhizogenes harboring CRISPR vector, establishing a gene editing vector detection system for cotton. Our result demonstrated that the A. rhizogenes-mediated method to induce hairy roots in cotton can be used to detect the target site mutation and lay the foundation for cotton genome editing and gene function veri cation.

Material And Methods
A work ow diagram for the experiments is given in Figure 4.

Plants material and growth conditions
Upland cotton cultivars Coker 312 (R15) and TM-1 were kept in our laboratory, Lumian 21 and Zhongmian 49 were bought from Beijing Shunheyuan Seed Co. LTD. These four cotton cultivars were cultured in a growth chamber (16-h light/8-h dark; 28 ℃; 70% humidity). Plants were used for inducing hairy root when the two cotyledons had fully expanded (about 10 days after sowing).

Construction of CRISPR/Cas9 vector
The human codon-optimized Cas9 gene (35S-Cas9-SK), driven by the CaMV 35S promoter and the chimeric single-guide RNA (AtU6-26-SK), driven by the AtU6-26 promoter were gifts from Jian-Kang Zhu. In this study, one single-guide RNA (sgRNA) in the second exon of GhMYB25-like gene was selected. A pair of DNA oligonucleotides was synthesized and annealed to generate a dimer. The dimer was then cloned into the BbsI site of pAt-U6-26-SK to generate pSK-At-U6-sgRNA. pSK-At-U6-sgRNA was cut with restriction enzyme KpnI/SalI, and 35S-Cas9-SK was digested with restriction enzyme SalI/EcoRI. Two fragments were assembled into pCAMBIA2301 using Kpn /EcoR restriction digestion followed by ligation to generate the p2301-GhMYB25-CRISPR construct. The kan gene, driven by the CaMV 35S promoter of pCAMBIA2301, was used as selection marker for cotton stable transformation. The positive plasmid was introduced into A. rhizogenes strain K599 to induce hairy roots.

Agrobacterium rhizogenes-mediated transient transformation of cotton
A. rhizogenes K599 harboring the p2301-GhMYB25-CRISPR construct was cultured at 28 ℃ with shaking (180 rpm) to different OD600s (0.4, 0.6, 0.8, 1.0). Then the apical meristem between the two cotyledons on 20 plants of cotton cv. R15 was injected with one of the concentrations. Plants were then grown in the growth chamber for 1 month, then the number of plants with hairy roots was counted. The other three cotton cultivars were then tested in the same way.
Genomic DNA extraction and mutations analyses One month after injection, any hairy roots produced were collected, and DNA was extracted using the DNAsecure Plant Kit (TIANGEN, Beijing, China). Primers U6-F/R designed based on the AtU6-sgRNA sequence to detect exogenous T-DNA, and primers VirD-F/R were designed based on the VirD gene to detect A. rhizogenes contamination (Additional le1: Table S1). The region spanning the target gene GhMYB25-like in the A genome and D genome was ampli ed using 2´ Taq Plus Master Mix (Vazyme, Nanjing, China) with the speci c primer pair (Additional le1: Table S1). The amplicons were sequenced using the platform HiTOM [14].

Statistical analyses
Samples were collected from more than three technical replicates for each injection. The number of plants with hairy roots data were analyzed using SPSS software (IBM, Armonk, NY, USA). Analysis of variance was used to compare the statistic difference based on Tukey's HSD test at signi cance level of p < 0.05.

Vector construction and target selection
The CRISPR vector encoded Cas9 driven by the CaMV35S promoter and one sgRNA driven by the Arabidopsis U6 promoter (Fig. 1a). Most genes in cotton are duplicated, so we sought to target GhMYB25like in both the A genome and D genome so that multiple homologues could be edited simultaneously. The sgRNA was designed to guide Cas9 to cleave the target site within the second exons of GhMYB25like in genomes A and D, and the primer site for ampli cation of region surrounding GhMYB25-like target site were also identical in the two different genomes (Fig. 1b).

Hairy root induction by different concentrations of A. rhizogenes
Among four concentrations of A. rhizogenes tested, OD600 = 0.6 and 0.8 induced more plants of cv. R15 to produce hairy roots than OD600 = 0.4 or 1.0 did (Fig. 2a). Otherwise Zhongmian 49, Lumian 21, TM-1 did not differ signi cantly from R15 when OD = 0.6-0.8 (Fig. 2b, c). It indicated that this method is suitable for cotton materials with different genetic backgrounds.

Identi cation of positive hairy root
In the test to verify the feasibility of the designed sgRNA using A. rhizogenes strain K599 with the CRISPR/Cas9 plasmid to induce hairy roots in cotton. Hairy roots had developed from the R15 injection position. Ten hairy roots lines were randomly selected and used for genomic DNA extraction. PCR analysis showed that all of these lines were co-transformed with the vector p2301-GhMYB25-like-CRISPR using the speci c primers (Fig. 3a), and virD was not ampli ed for any line, thus excluding A. rhizogenes contamination (Fig. 3b).

Con rmation of target site in cotton hairy roots
The region surrounding the target sequence in GhMYB25-like in the A and the D genome was ampli ed by PCR in 10 positive hairy root lines. Sequencing of the amplicons using Hi-TOM revealed mutations in the R15 hairy roots of lines L1, L2, L5, L6 and L10. Eight mutation types were found at the target site in genome A, including a 1-, 2-, 4-, 5-, 6-, 8-, and 9-bp deletion and a 1-bp insertion; seven mutation types were found in genome D, including a 1-, 3-, 4-, 5-, 6-, 7-, and 8-bp deletion (Fig. 3c). These results illustrate that the transgene-encoded Cas9 and sgRNA were able to e ciently induce double-strand breaks at the target site in GhMYB25-like in the A and the D genome and that the recombinant vector can be used for stable genetic transformation of cotton.

Discussion
The use of mutants is particularly important for studying gene functions and molecular mechanisms, and the CRISPR/Cas system provides new mutations and new methods to target genes for functional research and genetic improvement of crops. CRISPR/Cas9 technology has been successfully applied to many crop genome engineering, including cotton [15][16][17][18]. Toward using sgRNA in allotetraploid cotton genome, here we injected shoot apical meristems with A. rhizogenes harboring vector p2301-GhMYB25like-CRISPR to produce hairy roots to assess the e cacy of the sgRNA. One sgRNA, designed for the same genomic region in the GhMYB25-like gene in the A genome and D genome, was employed to examine the sgRNA e cacy [19,20]. The CRISPR/Cas9 system was used to construct a single-target vector to knock out the cotton GhMYB25-like gene. The best OD values for A. rhizogenes to use for the injection were 0.6 and 0.8 (Fig. 2a), and the method is suitable for cotton material with different genetic backgrounds ( Figure. 2b, c). After the apical meristem was injected, 10 seedings developed hairy roots, namely lines L1-L10 (Fig. 3a), were obtained and analyzed further. Among these 10, 5 mutant lines (L1, L2, L5, L6, L10) were edited at the target site. Our results indicate that this method is feasible for detecting the target site, operation is relatively simple, and the feasibility of selected target knockout site in the gene can be quickly determined.

Declarations Acknowledgments
We thank Jiankang Zhu for the CRISPR/Cas9 vector. We appreciate all the colleagues in our laboratory who provided experimental materials. The CRISPR vector encoded Cas9 driven by the CaMV35S promoter and one sgRNA driven by the Arabidopsis U6 promoter (Fig. 1a). Most genes in cotton are duplicated, so we sought to target GhMYB25like in both the A genome and D genome so that multiple homologues could be edited simultaneously.
The sgRNA was designed to guide Cas9 to cleave the target site within the second exons of GhMYB25like in genomes A and D, and the primer site for ampli cation of region surrounding GhMYB25-like target site were also identical in the two different genomes (Fig. 1b).