Design

1 Working principle of the project

CRISPR/Cas9 is a revolutionary gene editing tool that uses RNA to target and cut specific DNA sequences. The CRISPR system consists of a guide RNA (gRNA or sgRNA) that guides the Cas9 enzyme to the desired location on the genome, and an RNA-DNA complex that cleaves the DNA. This allows scientists to remove specific genes with incredible precision (Fig. 1)).

Fig.1 How CRISPR works (Ball P, 2016)

In plants, the biosynthesis of starch requires five classes of core enzymes, including ADP-glucose pyrophosphorylase (ADPG), starch synthases (SS), starch branching enzymes (SBEs), starch debranching enzymes (DBEs), and granule-bound starch synthase (GBSSI), that are located in the chloroplast or amyloplast (Tetlow IJ, 2006; Tian ZX, et al. 2009; Fig. 2). In sweet potato, GBSSI is responsible for amylose biosynthesis, and amylopectin is not only regulated by starch branching enzymes (SBEs) but also influenced by soluble starch synthase (SS) and debranching enzymes (DBEs) including isoamylase (ISA) and pullulanase (PUL) (James MG et al., 2003; Lyu RQ et al., 2021)

Fig.2 A simplified starch synthesis system in plants（Tian ZX et al., 2009）

Eighteen genes are involved in or play distinct roles in different steps of starch synthesis. AGP, ADP-glucose pyrophosphorylase; AGPlar, AGP large subunit; AGPiso, AGP large subunit isoform; AGPsma, AGP small subunit; GBSS, granule-bound starch synthase; SS, soluble starch synthase; SBE, starch branching enzyme; ISA, isoamylase; PUL, pullulanase; ISA and PUL belong to starch debranching enzyme (DBE)

Given the feasibilities such as technological intricacy and the Limited project duration, our current approach aims to knock out gene the GBSS gene in sweet potato.we can knock out the gene GBSS using CRISPR/Cas9 system, by using a sgRNA that target for the gene GBSSI, which can effectively block the pathway for the synthesis of amylose, so that ADP-glucose can only be synthesized into amylopectin

2 General design

By modifying the starch synthesis pathway of sweet potato to obtain high-purity amylopectin for the production of starch-based biodegradable plastics, the entire project can be divided into several missions (Fig. 3):

1. Vectors construction: to construct the backbone vector and expression vector of sgRNA (IbGBSSI)
2. Genetic transformation of sweet potato: to transform the expression vector of sgRNA (IbGBSSI) into sweet potato
3. Starch extraction and analysis: to extract and analyze the high-purity amylopectin from storage roots of sweet potato and
4. production of starch-based biodegradable plastics: to adjust the content ratio of amylopectin to amylose in raw starch to produce starch-based bioplastics

Fig. 3 Work flow chart of the project

3 Vectors construction

By referring to Liu WS 's (2016) procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana, we planned to construct the vector through the following steps.

3.1 sgRNA target Selection and sgRNA oligo synthesis

The appropriate sgRNA targets for the gene IbGBSSI (Accession Number: AB071604) will be selected using the online sgRNA design tools. The following are some web-based sites for the selection of sgRNA targets:

1. http://www.plantsignal.cn/CRISPR/crispr_primer_designer.html
2. http://www.genome-engineering.org/crispr/?page_id=41
3. https://www.dna20.com/eCommerce/cas9/input
4. http://www.genome.arizona.edu/crispr/index.html

Once a 20-nt sgRNA target site is selected, a pair of sgRNA oligos will be obtained by annealing later.

3.2 Construction of the backbone vector

The plasmid psgR-Cas9-At will be is digested and the products will be ligated with sgRNA oligos to obtained a sgRNA-Cas9 plasmid, the backbone vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI). In the backbone vector of sgRNA (IbGBSSI), the sgRNA-Cas9 cassette should contain the single gRNA cassette with the Cas9 endonuclease, the U6 promoter from Arabidopsis (pAtU6) and the Arabidopsis ubiquitin-1 promoter (AtUBQ1). pAtU6 will be used to drive the sgRNA expression and the AtUBQ1) will be used to control Cas9 expression (Fig. 3).

The ligated DNA will be then transformed into bacteria. The obtained clones will be validated by PCR later.

3.3 Construction of the plant expression vector

The validated backbone vector will be digested and inserted into the binary vector pCAMBIA1301s, harbouring the Hygromycin B resistance gene HygR and the reporting gene GUS, to obtain the plant expression vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI)-p1301s. PCR will be used for its verification(Fig.4).

Fig. 4 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.

The expression vector of sgRNA (IbGBSSI) will be transferred into Agrobacterium tumefaciens LB4404 by freeze-thaw method. The positive transformants will be selected with antibiotics of rifampicin, chloramphenicol and kanamycin during incubation and validated by PCR later

4 Genetic transformation of sweet potato

4.1 Callus induction of sweet potato

According to Yang (2011), genetic transformation of sweet potato will be performen on embryogenic calli. Therefore, embryogenic calli must be induced in advance from axillary buds of pre-prepared sterile sweet potato seedlings, and proliferated through solid culture and suspension culture

4.2 Agrobacterium-mediated transformation

When enough of sweet potato embryogenic calli are obtained, they will be infected with the A. tumefaciens transformants containing the vector for CRISPR-knockout expression by co-culture. Positive transformed calli will be screened through culture with hygromycin

4.3 Culture and verification of mutant seedlings

The positive transformed calli were further cultured to obtain transgenic sweet potato seedlings, which will be validated using GUS test of leaves at first. During GUS testing, if leaves of a seedling turn blue, the seedling may be a mutant. A further verification will be also performed by PCR with primer pairs of the genes Cas9 and Hyg.

Later, the positive transformed lines of sweet potato will be planted using the method of cuttage in an experimental greenhouse to harvest starch-rich tubers. During the vegetative growth of the plants, chlorophyll detection will be carried out to observe the effects of gene-knockout on plant’s growth physiology. Meanwhile, the gene relative expression level of IbGBSSI in storage roots will be detected by Quantitative-PCR

5 Starch extraction and analysis

After three months of transplantation, the sweet potato storage roots will become large enough and accumulate sufficient starch. At this time, the storage roots will be collected and cleaned, and the total starch will be extracted

Qualitative detection of the starch components will be carried out by iodine staining to see whether the starch contains amylose, as amylose turns blue while amylopectin appears reddish-brown or purple-red when exposed to iodine

Quantitative detection of the starch composition will be performed using the method of spectrophotometry (NY/T 2639-2014). The OD620 values of total starch solutions will be determined. And then the OD620 values will be plugged into the standard curve for amylose content of total starch to quantify the amylose content of each sample

6 Production of starch-based biodegradable plastics

In the future, after the engineered sweet potato varieties are widely cultivated, a significant amount of high-purity amylopectin can be obtained. This pure amylopectin can be used directly as the primary raw materials for starch-based biodegradable plastics without additional separation and purification processes, and the content ratio of amylopectin to amylose in the total starch raw materials can be adjusted by increasing or decreasing the amount of the pure amylopectin to produce starch-based biodegradable plastics with different performance: For example, the tensile strength of the product can be increased by reducing the proportion of amylopectin, and by increasing the proportion of amylopectin, the tensile performance of the product can be enhanced.

7 Reference:

Ball P. (2016) CRISPR: Implications for materials science. MRS Bulletin, 41: 832-834.

James MG， Denyer K， Myers AM. (2003) Starch synthesis in the cereal endosperm. Curr. Opin. Plant Biol., 6: 215–222.

Liu WS, Zhu XH, Lei MG et al. (2015) A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana. Sci. Bull., 60(15): 1332–1347.

Lyu RQ, Ahmed S, Fan WJ. (2021) Engineering properties of sweet potato starch for industrial applications by biotechnological techniques including genome editing. Int. J. Mol. Sci., 22(17): 9533.

NY/T 2639-2014. Determination of amylose content in rice - Spectrophotometry method.Tian Z, Qian Q, Liu Q et al. (2009) Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities. PNAS, 106 (51): 21760-21765

Tetlow IJ. (2006) Understanding storage starch biosynthesis in plants: A means to quality improvement. Can. J. Bot., 84: 1167–1185.

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.