Engineering Success

1 Vector construction

1.1 Design and synthesis of sgRNA

The sweet potato granule-bound starch synthase I (IbGBSSI) gene (Accession Number: AB071604) contains nine exons and CRISPR/Cas9 targets the first one. The appropriate sgRNA targets for the gene IbGBSSI were selected using the online sgRNA design tools and the available I. batatas draft genome sequences (Yang et al., 2017). The selected sgRNA is located 70 bp downstream of the start codon, with a total sequence length of 27 bp (Fig. 1). (The selected sgRNA will be cloned from the target gene.)

sgRNA oligos were obtained by annealing with the designed primers following:

Oligo 1 (GBSSI-sgRNA1): 5’-GTGGGGTTGGGTCAATTAGCCCTGAGGAGC-3'

Oligo 1 (GBSSI-sgRNA2): 5’-AACTGGTGGACTTGGAGATGTTCTTGGAGG-3’

Fig. 1 The target region of gene IbGBSSI and the location of the gRNA

Exons are shown as square frames and surrounding introns appear as lines. sgRNA and PAM are highlighted in yellow and green, respectively.

1.2 Construction of the backbone vector

The plasmid psgR-Cas9-At were digested (Fig. 2) and the products were ligated with sgRNA oligos using T4 ligase to obtained the backbone vector of sgRNA (IbGBSSI): psgR-Cas9- sgRNA(IbGBSSI). 1/2 of the DNA was then transform into bacteria and incubate in LB for 45 min, and was plated in LB containing Ampicillin (Fig. 3). The obtained clones were validated by PCR with primers M13F/oligo2 (The sequence of M11F was: 5’-TGTAAAACGA CGGCCAGT-3’) (Fig. 4).

Fig. 2 Digestion result of psgR-Cas9-At

Fig. 3 E. Coli transformed with backbone vector of sgRNA (IbGBSSI)

Fig. 4 PCR product of sgRNA (IbGBSSI)

1.3 Construction of the expression vector

The validated backbone vector of sgRNA (IbGBSSI) was digested by EcoR I & Hind III (Fig. 5) and inserted into the corresponding sites of the binary vector pCAMBIA1301s, harbouring the Hygromycin B resistance gene HygR and the reporting gene GUS, to obtain the expression vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI)-p1301s.

Fig. 5 Digestion result of psgR-Cas9-sgRNA and pCAMBIA1301s

The expression vector of sgRNA (IbGBSSI), harbouring the single gRNA cassette with the Cas9 endonuclease, were constructed. The U6 promoter from Arabidopsis (pAtU6), was used to drive the sgRNA expression. The Arabidopsis ubiquitin-1 promoter (AtUBQ1) was used to control Cas9 expression (Fig. 6).

Fig. 6 CRISPR/Cas9 gene editing construct for the target gene IbGBSSI.

Structural organization of the CRISPR/Cas9 binary vector pCAMBIA1301s used for stable Agrobacterium-mediated transformation in the sweet potato. A. thaliana promoter AtU6 drives expression of the sgRNA. The cauliflower mosaic virus promoter (CaMV 35S) drives expression of the Cas9 gene. Abbreviations: NLS, nuclear localization signal; Nos, Nos terminator.

1.4 Agrobacterium tumefaciens transformation

The expression vector of sgRNA (IbGBSSI) was successfully transferred into Agrobacterium tumefaciens LB4404 by freeze-thaw method. The positive transformants containing were selected and cultured on solid YEB medium plates containing antibiotics of rifampicin, chloramphenicol and kanamycin (Fig. 7). The selected clones were validated by PCR later with primers M13F/oligo2 (Fig. 8).

Fig. 7 A. tumefaciens transformed with the vector psgR-Cas9-sgRNA(IbGBSSI)-p1301s.

Fig. 8 PCR result of A. tumefaciens transformed with psgR-Cas9-sgRNA(IbGBSSI)-p1301s

2 Genetic transformation of sweet potato

The stable transformation of sweet potato was performed using the protocol of Yang et al (2011) .

2.1 Induction and culture of embryogenic callus

We took the axillary buds from the pre-prepared sterile sweet potato variety B23 seedlings, inoculated the meristematic tissue onto MSD solid medium, and successfully induced embryogenic callus (Fig. 9). Afterwards, these embryogenic calli were transferred to new MSD solid medium and LCP liquid medium successively for propagation (Fig 10).

Fig. 9 The process of sweet potato embryogenic callus induction

Fig. 10 Embryogenic callus culture of sweet potato

2.2 Agrobacterium-mediated transformation

When enough of sweet potato embryogenic callus ware obtained, they were infected with the A. tumefaciens transformants containing the vector for CRISPR-knockout expression by co-culture (Fig. 11A). After selection with hygromycin, positive transformed calli were obtained (Fig. 11B). These calli were further cultured to obtain transgenic sweet potato seedlings (Fig. 12).

Fig. 11 Embryogenic callus co-culture with A. tumefaciens transformants (A) and selection of postively transformed callus with hygromycin (B)

Fig. 12 Seedlings of IbGBSSI-knockout lines after preliminary selections

3 Verification of transgenic sweet potato plants

3.1 GUS detection

he GUS gene, is a commonly used reporter gene harboured in pCAMBIA1301s. Its expression product β-glucuronidase is a hydrolase that can catalyze the hydrolysis of many β-glucoside esters. It can decompose X-Gluc into blue substances, to observe the expression of foreign genes in transgenic plants and identify transgenic plants.

After the regenerated seedlings grew leaves, we conducted GUS staining on these transgenic plants and preliminarily screened two successfully transformed sweet potato lines, designated as 23216004 and 23216005 (Fig. 13).

Fig. 13 Results of GUS staining

3.2 PCR detection

Genome DNA of these two transgenic lines was extracted from leaves of these two regenerated seedlings. PCR detection on the genomes was performed by using two pairs of primers which were designed based on the Cas9 protein gene and the hygromycin resistance gene (HygR), respectively. As the result, the transgenic lines 23216004 and 23216005 were further validated (Fig. 14).

The sequences of the two pairs of primers were as bellow:

• CAS9-F: 5’-ATGGACTATAAGGACCACGACGG-3’;
• CAS9-R: 5’-TTGTCGCCTCCCAGCTGAGACAG-3’
• HygR-F: 5’-Atgaaaaagcctgaactcac-3’;
• HygR-R: 5’-ctatttctttgccctcggac-3’

Subsequently, these two transgenic lines and the wild-type B23 were planted with the method of cuttage in an experimental greenhouse to harvest starch-rich tubers.

Fig. 14 Detection results of PCRs for gene Hyg and gene Cas9

3.3 Determination of chlorophyll content in leaves

During the cultivation process, there is no visible difference in the above-ground phenotype between the transgenic line and the wild type. To confirm whether the gene knockout has an impact on the physiology of the transgenic line, we measured the chlorophyll content in their leaves.

Each transgenic line and wild type were sampled from three different plants, and the samples were prepared into chlorophyll solutions. The absorbance values of the solutions at wavelengths of 663 nm and 645 nm were determined using spectrophotometry method, designated as OD663 and OD660, respectively. Afterwards, the following formulas were applied to calculate the concentrations of chlorophyll a, chlorophyll b, and total chlorophyll for each sample:

Chl a = 12.7 x OD663 - 2.69 x OD646 (1)

Chl b = 22.9 x OD646 - 4.68 x OD663 (2)

Chl TOT = 8.02 x OD663 + 20.2 x OD646 (3)

In the formulas,

Chl a: Content of chlorophyll a (ug/L);

Chl b: Content of chlorophyll b (ug/L);

Chl TOT: Total content of chlorophyll a and chlorophyll b (ug/L);

According to the results, it could be seen that both transgenic lines have chlorophyll A and B contents similar to the wild type, at around 0.6000 mg/g and 0.2500 mg/g respectively (Table 1; Fig. 15). It showed that the chlorophyll contents of the transgenic lines were basically consistent with that of the wild type, ruling out the impact of gene-knockout on the sweet potato starch synthesis pathway.

Table 1 chlorophyll contents of the transgenic lines

Line No.	mass	OD663	OD645	congtent of chl a (mg/g)		congtent of chl b (mg/g)		congtent of total chl (mg/g)
					mean+SD		mean+SD		mean+SD
B23	spl1	0.4722	2.6045	1.0812	0.6389	0.6085+0.0304	0.2662	0.2535+0.0127	0.9186	0.8749+0.0437
	spl2	0.4571	2.4012	0.9968	0.6085		0.2535		0.8749
	spl3	0.4623	2.3071	0.9577	0.5781		0.2408		0.8311
23216004	spl1	0.4812	2.6173	1.0909	0.6298	0.5998+0.0300	0.2646	0.2520+0.0126	0.9078	0.8646+0.0432
	spl2	0.4931	2.4266	1.0115	0.5698		0.2394		0.8213
	spl3	0.4786	2.4792	1.0334	0.5998		0.2520		0.8646
23216005	spl1	0.4802	2.6184	1.0828	0.6318	0.6018+0.0301	0.2612	0.2487+0.0124	0.9063	0.8631+0.0432
	spl2	0.4922	2.4282	1.0041	0.5717		0.2363		0.8200
	spl3	0.4768	2.4760	1.0239	0.6018		0.2487		0.8631

chlorophyll contents of the transgenic lines

3.4 Determination of the expression level of IbGBSSI in storage roots

Two months after transplantation, the storage roots of the transgenic lines were harvested. It showed that the number and size of the root tubers were largely consistent with the wild type (Fig. 16).

Fig. 16 Phenotypes of the IbGBSSI-knockout lines planted in greenhouse

RNA was extracted from the fresh root tubers and cDNA was obtained through reverse transcription later. Then, the relative expression level of the gene IbGBSSI was determined with the method of Quantitative Real-time PCR with the root-tuber cDNA as templates. The result showed that the relative expression level of IbGBSSI in storage roots of the transgenic lines (0.1063 and 0.2407) was much lower than that of the wild type (1.0000) (Fig. 17). It revealed that the knock-out of IbGBSSI in the pathway of starch synthesis was successful.

Fig. 17 Q-PCR result of the relative expression level of IbGBSSI in root tubers

4 Starch analysis of transgenic sweet potato root tubers

Freshly harvested sweet potato tubers were cleaned, peeled, and sliced into small pieces. Starch was extracted from these pieces for qualitative and quantitative detection afterwards.

4.1 Qualitative detection of the starch components

When exposed to iodine, amylose appears blue, while amylopectin appears reddish brown or purple red. Therefore, the component qualitative detection of the total starch from the transgenic lines was performed. As the result, the total starch of the transgenic lines appears reddish brown while that appears blue of the wild type (Fig. 18). It indicated that the total starch of the transgenic lines was composed mainly of amylopectin.

Fig. 18 Component detection of total starch by iodine staining

4.2 Quantitative detection of the starch composition

Firstly, standard starch solutions with different gradients of amylose content were prepared (Table 2) and their absorbance values at a wavelength of 620nm, OD620, were obtained (NY/T 2639-2014). Subsequently, using the absorbance values as the vertical axis and the amylose content as the horizontal axis, the standard curve representing the linear relationship was plotted between the content of branch starch and OD620 and the following calculation formula was fitted (where y represents the content of amylose in total starch (%), and x represents OD620) (Fig. 19):

y = 197.47x - 14.829, R² = 0.9977 (4)

Table 2 OD620 values of standard solutions of starch

OD620	Content of amylose (%)
0.092	0
0.126	10
0.170	20
0.219	30
0.272	40
0.377	60
0.480	80
0.589	100

Fig. 19 Standard curve for amylose content (%) of total starch

Later, starch solutions were prepared respectively from total starch extracted earlier from storage roots of the two genotypes and the wild type and their OD620 values were determined, with three replicates for each line. After plugging the obtained OD620 values into formula (4), the amylose content was quantified in each sample.

Compared to the amylose content of the wild type (23.3306%), the amylose content in the total starch of the two transgenic lines was only 0.5657% and 0.7613%, respectively (Table 3, Fig. 20). According to the study by Wang (2019), knockout of IbGBSSI had no impact on the total starch content in sweet potato tubers. It indicated that sweet potato lines of high-content amylopectin synthesis have been developed with the knock-out of the gene IbGBSSI by using a highly efficient CRISPR/Cas9 system.

Table 3 Amylose content (%) of total starch of root tubers

Line No.	Mass (mg)	OD620	Content of amylose (%)
				Mean+SD
B23	spl1	10.21	0.1970	23.5775	23.3306+2.4801
	spl2	9.99	0.2050	25.6780
	spl3	9.99	0.1800	20.7363
23216004	spl1	10.10	0.0790	0.7635	0.5657 +0.3367
	spl2	10.10	0.0760	0.1770
	spl3	10.19	0.0790	0.7568
23216005	spl1	10.26	0.0790	0.7516	0.7613 +0.4862
	spl2	9.91	0.0765	0.2800
	spl3	10.10	0.0815	1.2523

Fig. 20 Amylose content (%) of total starch of root tubers

Future work

Our long-term goal is to develop multiple types of high-performance biodegradable plastics using starch as raw material. Due to the differences in physical and chemical properties between amylose and amylopectin, different ratios of amylose to amylopectin in raw starch can lead to different properties of starch-based bioplastics. Therefore, by adjusting the ratio of amylose and amylopectin in raw starch, we can obtain bioplastics with different properties to meet the needs of society and the pure amylose and pure amylopectin will be undoubtedly the most suitable. However, the existing starch separation and purification technologies usually involve complex processes and expensive equipment. Obtaining high-pure amylose and high-pure amylopectin with industrial methods will result in high production costs for starch-based bioplastics. It is obviously there is a great demand for natural high-pure amylose and high-pure amylopectin.

Therefore, in subsequent related research, we will also obtain sweet potato lines with high-amylose synthesis through similar gene editing techniques. Additionally, based on the obtained sweet potato lines with high-amylopectin synthesis and high-amylose synthesis, we will seek to further enhance the proportion of amylose or amylopectin in these plants through other biotechnological means so that we can extract the high-pure amylose or high-pure amylopectin directly from sweet potato root tubers. This will eliminate the starch separation and purification process required for the preparation of starch-based bioplastics, thereby reducing their production cost greatly and promoting the application of starch-based biodegradable plastics well.

Reference

Wang HX, Wu YL, Zhang YD et al. CRISPR/Cas9-based mutagenesis of starch biosynthetic genes in sweet potato (ipomoea batatas) for the improvement of starch quality. Int. J. Mol. Sci. 2019, 20, 4702; doi:10.3390/ijms20194702

Yang J, Bi HP, Fan WJ et al. Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.). Plant. Sci. 2011, 181, 701–711.

Yang J, Moeinzadeh MH, Kuhl H et al. Haplotype-resolved sweet potato genome traces back its hexaploidization history. Nat. Plants 2017, 3, 696–703.

NY/T 2639-2014. Determination of amylose content in rice - Spectrophotometry method