• overview

During the process of the project, we have carried out several safety assessments through human practice activities and exchanges and cooperation, and constantly iterated the experimental technology. We can ensure that our engineered organisms will not be released into the environment and cause harm to the environment and human body in the process of the project, and we are fully prepared for the application of future projects to production. We will strictly follow the requirements of China's gene editing regulations to complete the approval process, and we have designed molecular markers and suicide switches to be applied in the next generation of technology iterations to ensure that our engineered organisms are easy to monitor and control when released.

• Laboratory safety

• Laboratory space:

After reviewing the classification of organism risk groups, we have confirmed that all the organisms we used were in risk group 1, so it is safe for us to conduct experimental operations on the open workbench and ultra clean workbench and in the tissue culture room. The laboratory is equipped with necessary safety equipment, such as eye washer, safety shower, fire equipment and first-aid kit.

• Laboratory safety training:

In April, our college organized all members of the iGEM wet team to carry out professional biological laboratory safety training to understand the operating procedures and potential risks of the safety laboratory, and our PIs will carry out safety training in the plant laboratory for us at each regular meeting of the team. During the specific experiment, senior graduate students will ensure the safety of the experimental details.

• Protective measures for laboratory personnel:

1. we have worn appropriate personal protective equipment in the laboratory, including laboratory clothes, gloves, masks, etc., to prevent direct contact of plant materials with the skin. When using liquid nitrogen, we have worn thickened cotton gloves to prevent frostbite.
2. when conducting experiments involving microorganisms, we have followed the principle of aseptic technology to avoid cross contamination.
3. the laboratory has well functioning ventilation facilities, including ventilation fans, convection doors and windows, and air conditioners, which can ensure air circulation. When using microbial products, we did it in the ultra clean workbench, and when using toxic volatile liquids, we did it in the fume hood.
4. after the experiment, we have sorted out, clean and disinfect the experimental area according to the specified procedures, and sterilize the microbial preparations.
5. before leaving the laboratory, we have token off our protective equipment, recycle it into the trash can dedicated to laboratory waste, and clean our hands.
6. our laboratory is equipped with emergency treatment measures. In case of accidental exposure or pollution, we have reported it to the tutor and the college for treatment at the first time. The exposed laboratory personnel will seek medical treatment immediately, and carry out laboratory cleaning and killing work.

• Experimental waste treatment measures:

1. for waste liquid, we have wasted liquid barrels with inner seals for collection, and the waste liquid is regularly taken away by relevant professionals for harmless treatment.
2. for solid wastes such as masks, gloves, plastic packaging, paper products, rice materials not contaminated by microbial agents and used gun heads (not used in experiments involving microorganisms), we have generally discarded them in the laboratory trash can dedicated to the laboratory and regularly take them away for cleaning.
3. for the waste such as culture medium, culture medium and gun head used in transformation experiments that have been contaminated by microorganisms, we have usually sterilized them first and then discard them in the laboratory trash can.

• Protective measures for dangerous reagents and chemicals:

(all the following hazardous chemicals will be collected in the waste liquid tank and regularly handed over to the special agency for treatment according to the regulations on hazardous waste treatment)
1. acids: we have ensured to wear protective clothing, gloves and goggles before use. Acidic waste liquid is generally neutralized to a pH value of 6-8 and collected in the waste liquid tank. 2. carcinogens: we have generally worn protective clothing, gloves, goggles, and operate in the fume hood. We have used a pipette gun to avoid splashing, contact, and inhalation. The waste liquid is collected in the waste liquid barrel.
3. organic solvents: we have generally operated with fume hoods and wear protective clothing and gloves to avoid inhalation of vapor or contact with skin.
4. heavy metal salt: we have worn protective clothing, gloves and goggles during operation to avoid intake and inhalation.
5. biotoxins: we have worn protective clothing, gloves, goggles and masks during operation, and collect them in the waste liquid tank after high-temperature inactivation treatment.

• Project Safety

• Organism:

Our chassis organism is rice, which is an organism on the white list, and we have consulted the safety and security committee by email before. They reminded us that we also need to fill in the Check In Form for engineering design in a multicellular biological system. We ensure that the Check In Form is completed synchronously before the deadline of the safety form.

• Parts

• For their information, see Wet-Lab——Parts

Parts in chassis
Including OsGluB-1 and OsGlb.
They are not in the Red Flag. They are plant protein coding genes on the white list, and they are glutelin and globulin encoded by rice endogenous genes. They are components of rice endosperm storage proteins and have no toxic effect on human body.
Partson multi-target CRISPR/Cas9 carrier for site directed editing
Including OsGluB1-sgRNA expression cassette, OsGlb-sgRNA expression cassette and Cas9 protein expression cassette
The OsGluB1-sgRNA expression cassette includes the following parts:
Backbone (pUC18), sgRNA of target OsGluB-1, snRNA promoter (AtU3b,AtU3d,AtU6-1,AtU6-29), Restriction sites (BamH-Ⅰ, Bsa Ⅰ(1), Bsa Ⅰ(2) , Hind Ⅲ), marker genes (LacZ, ApR), primers (U-F/gR-R, Pps/Pgs)
The OsGlb-sgRNA expression cassette includes the following parts:
Backbone (pUC18), sgRNA of target OsGlb, snRNA promoter (AtU3b，AtU3d，AtU6-1，AtU6-29）, Restriction sites (BamH-Ⅰ, Bsa Ⅰ(1), Bsa Ⅰ(2) , Hind Ⅲ), marker genes (LacZ, ApR), primers (U-F/gR-R, Pps/Pgs)
Cas9 protein expression cassette includes the following parts:
Cas9p and its promoter PUbi, marker genes (HPT, Bar), their promoter 2xP35S, promoter T35S, polyadenylation signal sequence aadA(KanR), replication origin pBR322, enhancer pVS1 replication, marker gene ccdB, left and right boundaries (LB, RB), left and right arm sequences (GA-L, GA-R) that guide sgRNA expression, restriction enzymes (Asc I, Not I), digestion sites Bsa I(B-L), Bsa I(B-R)

The above parts have no toxic effect on plants or animals, and the snRNA promoter, marker gene, Cas9 protein gene with optimized codon and its promoters PUbi, aadA, enhancer pVS1 replication, restriction enzyme, GA-L, GA-R can reduce the safety risk of off target due to gene editing.
However, individual parts may increase the risk of horizontal gene transfer. The corresponding parts and reasons are as follows. We have register them in Check In Form.
1. Replication origin pBR322: this part allows plasmids to replicate in bacteria. If plasmids enter bacteria in the environment, these bacteria may replicate and transmit these plasmids. See Agrobacterium residues ,Transportfor details of management risks.
2. Enhancer pVS1 replication: enhancers can improve the transcriptional activity of genes, which may increase the expression level of genes in bacteria. See Agrobacterium residues ,Transportfor details of management risks.
3. Marker gene ccdB: This is a toxic gene that can kill bacterial cells without the plasmid, which may lead to the selective advantage of bacteria containing the plasmid in the environment. See Protective measures for dangerous reagents and chemicals: , Agrobacterium residues ,Transportfor details of management risks.
4. Left right boundary (LB, RB): These are the boundary sequences of T-DNA, which can promote the insertion of T-DNA into the plant genome. If these sequences are transferred to other organisms, they may promote the gene insertion of non target organisms.
5. Left and right arm sequences (GA-L, GA-R) that guide sgRNA expression: these sequences contribute to the correct expression and processing of sgRNA. If they are transferred to other organisms, they may affect the gene regulation mechanism of those organisms.
6. Restriction enzymes (Asc I, Not I): these enzymes are used to cut DNA at specific positions. If these enzymes are transferred to other organisms, they may promote non-specific DNA recombination events.
7. Enzyme digestion sites Bsa I(B-L), Bsa I(B-R): these enzyme digestion sites allow the cleavage of DNA at specific positions. If these sites are transferred to other organisms, it may promote non-specific DNA recombination events.
See Transformation vector residue for management risks of 4-7

• Activities:

The engineering design activities of our experiment on rice include two, using colchicine to induce rice genome doubling and using CRISPR/Cas9 gene editing technology to carry out site directed mutagenesis on the two genes.
There may be several colchicine residues, Agrobacterium residues and transformation vector residues in the risk concerns of the activities. We have taken the following measures to manage these risks.
Colchicine residue:
1. through many attempts and explorations, we selected the appropriate colchicine treatment time to ensure that the concentration of colchicine in the callus can effectively induce chromosome doubling without excessive accumulation leading to residue.
2. after colchicine treatment, we put the callus in colchicine free medium for recovery culture, so that the colchicine in the body can be naturally metabolized and degraded.
3. after the polyploidization treatment, we cleaned the callus and changed the medium for many times to reduce the residue of colchicine in plant tissue.
4. during plant growth, the residual amount of colchicine should be regularly detected to ensure that it is within the safe range.
Agrobacterium residues:
1. optimizing the transformation process: in the transformation process, we found the appropriate amount of strains and culture conditions through adjustment and optimization to ensure the transformation efficiency and reduce the residue of strains.
2. The agrobacterium strain we used was EHA105, so gentamicin agrobacterium sensitive to was used to remove residual agrobacterium.
Transformation vector residue:
1. screening after culture: after transformation, hygromycin is used for screening to ensure that only the target gene is contained in the plant cells, but not the vector sequence. Because the function of our CRISPR/Cas9 transformation vector is to cleave the target gene rather than integrate into the target gene, the transformed cells themselves do not have hygromycin resistance, so hygromycin can screen out the cells with residual vector.
2. strict detection: when we identify mutants, we can confirm that there is no vector residue in and near the target gene. In order to further ensure safety, before commercialization, we will carry out more stringent genome-wide detection to ensure that there is no vector residue in rice.

• Release:

In the early stage, we were not sure whether our engineering rice planting in a pilot field of a research institute cooperating with us was a release behavior beyond containment. We have received a reply from the iGEM Safety Committee. They told us that this was not an excess volume release. Therefore, we can ensure that we do not intend to make any action to release genetically modified organisms into the environment during the project process, and we focus on better laboratory results.

• transport:

In the process of the project, we have cooperated with several fixed detection companies and primer synthesis companies to transport our PCR products, primers, and engineered rice seeds that can pass the 80 mesh screen in Wuhan. On the other hand, we also obtained an empty expression vector for CRISPR/Cas9 gene editing from our collaborative research group at South China Agricultural University.
For the transportation of these biological products, we have chosen SF Express. In order to ensure transportation safety, we have strictly followed the packaging requirements of the courier company.
1、 Selection of Packaging Materials
Special packaging bags: We have used SF Express to provide professional packaging bags for genetic testing samples, which usually have good sealing and leak proof properties to prevent samples from leaking during transportation.
Shockproof materials: We have used appropriate shockproof materials inside the packaging bag, including foam box and bubble film. Wrap the sample to prevent damage during transportation due to vibration or collision.
Outer packaging: We will put the specialized packaging containing insulated cotton with buffering effect into the express box, which has sufficient strength and hardness to withstand compression and impact during transportation.
2、 Packaging process requirements
Check sample bag: Before packaging, we have checked the sample bag for completeness and ensure there is no risk of leakage.
Fill in the information: We have filled in the personal information and sample information as required for accurate pickup and receipt.
Labeling: Delivery personnel usually paste the shipping label provided by the testing agency in a prominent position on the delivery box in front of us to ensure accurate delivery.
Sealed packaging: When the courier receives the package, both parties will ensure that all packaging materials are properly sealed to prevent contamination of the sample during transportation.

• Product Safety

Our engineered rice only doubled and mutated the endogenous genes, and did not introduce foreign genes. According to the current laws and regulations in China, it is differentiated from transgenic rice to supervise and evaluate the safety. Because we have focused on laboratory results during the competition and did not conduct any animal experiments and production tests and applications, we could not obtain accurate data other than genetic stability, environmental safety and food safety. In the future, we have submitted more complete target genes, gene editing methods, target gene editing, vector sequence residues, off target conditions, genetic stability, environmental safety, food safety and apply for production and application safety certificates according to the relevant laws and regulations of gene editing crops in China, and then make it a product.

The risk concerns about product safety mainly will come from the following aspects: polyploid and diploid hybrid sterility, and gene pollution of gene editing crops. These two problems are mainly caused by pollen dissipation. Therefore, we have mainly used the following two methods to manage the risks of future production applications: designing molecular markers to make our engineered rice more easily distinguished from conventional hybrid rice, and designing suicide switches to limit pollen dissipation.
Molecular markers:
rice proteins are differentially expressed in various organs, so we can change the color of rice husk by CRISPR/Cas9 gene editing technology to realize that rice seeds and regulatory agencies can visually distinguish our engineered rice and ordinary rice by the color of rice seeds. We uphold a responsible attitude, do not evade supervision, do not cheat, and protect the right to know of seed consumer groups.
The inheritance of rice glume color is controlled by both single gene and 2-3 pairs of interacting genes. In order to simplify the genetic operation on the premise of achieving the goal, we choose the strategy of single gene control. The golden yellow glume control gene OsCHI is a gene that can independently control the color of rice glumes. OsCHI encodes a chalcone isomerase, which can catalyze the isomerization of naringin chalcone to form biologically active dihydroxyflavanone. It is a key step in the pigment synthesis pathway to convert the intermediate chalcone into the final product anthocyanins (including anthocyanins, peony pigments, Pelargonium pigments, morning glory pigments, clove pigments, mallow pigments) and other water-soluble pigments. Moreover, the lack of expression of OsCHI gene only causes the deepening of the color of rice seed glume and internode, and does not change the flowering date, yield, rice quality and other major Rice Agronomic shapes.

Fig.1 Illustration of the anabolic pathways of rice anthocyanins Pelargonium pigment and cyanidin / paeoniflorin

There are mainly two kinds of anthocyanins in Rice: cyanidin-3-O-glucoside (cyanidin) and paeoniflorin-3-O-glucoside (paeoniflorin). CHI, as a key enzyme in the pigment synthesis pathway of Pelargonium, a non major anthocyanin species, is knocked down by CRSIPR/Cas9 gene editing technology, resulting in the upregulation of the expression of rice anthocyanin early biosynthetic gene (EBG) OsCHS and late anthocyanin biosynthetic gene (LBG) OsANS. ANS is a key enzyme in the late synthesis of Pelargonium pigment and cyanidin synthesis pathway. Because the Pelargonium pigment synthesis pathway is blocked by OsCHI knockdown, the Cyanidium / Paeonia pigment synthesis pathway is strengthened.
Therefore, we will chose OsCHI as the target gene. The following is the basic information of OsCHI, which comes from UniProt database.

Fig. 2 Gene information of OsCHI

Suicide switch:
since the main form of rice pollination is self pollination, we only need to prevent uncontrolled pollen escape from cross pollination.
This will be the design of our future work.
Our first design scheme will introduce a male gamete inactivating gene ZmAA1 and an inducible promoter into the original CRISPR/Cas9 vector, predict the difference of environmental physical conditions such as temperature, humidity and pressure between the two conditions when the pollen is in the bud and in the air through further modeling, select a factor with the largest difference as the response factor, start the expression of suicide genes, and make the scattered Pollen Sterile, while self pollination is not affected.
However, after further reviewing the literature, we found that the cause of death of ZmAA1 is not its own, but its mutation. The simple use of inducible promoter may not be able to accurately start and close the lethal mechanism, and the inducible promoter of environmental factors tends to have regulatory errors due to local climate change, which will lead to product safety and production. So the team leader consulted Zhenghui Lu, the lecturer of the biotransformation technology course she was studying, with questions. He proposed a new idea for us. See HP-Integrated Human Practices for details.
Our second design will consider changing to specifically express a toxic protein that only affects pollen fertility in rice pollen, and express an antitoxic protein that antagonizes this toxic protein in pistil stigma. In this way, self pollination, as the main breeding method, will not be affected, and the scattered pollen will be sterile due to the loss of antagonism, which can block the gene transmission of engineered rice across varieties.
Guided by this inspiration, we found the first antagonistic combination: AGL80 and MIKC* MADS-box transcription factors such as AGL62 and AGL65, which affect pollen fertility by competitively binding to specific gene promoters of accessory cells and antipodal cells in central cells. Unfortunately, the tissue specificity of AGL80 is not pollen, and transcription factors are proteins in the nucleus, so it is difficult to achieve the regulation of one cell to another by using them as targets.
When looking for the target again, we considered: since it is the mutual recognition of two cells, isn't it more direct to use the interaction of receptor and ligand to regulate the recognition process of pollen and stigma? Therefore, we noticed two mechanisms of rice stigma recognizing pollen homology. One is the S-locus genes, which encode the female determinant SCR on stigma and the male determinant SRK on pollen. The other is the stigma reactive oxygen species level regulated by the competitive binding of pollen PCP-B small peptide(membrane protein) and stigma RALP33 small peptide(secretory protein) with stigma FER/ANJ receptor kinase. Both of them can judge the "me" and "non me" of pollen. Using this "me" and "non me" recognition mechanism, we can make the pollen "non me", and the stigma can restore it to "mine", so that the scattered pollen can not be recognized by wild rice, and self pollination is not affected. (this mechanism itself has been a line of defense for engineered rice genes to drift to other species)
We compared the two mechanisms and found that the first mechanism is difficult to be considered reprogramming. Therefore, first of all, we need to turn pollen into a "non self" state. We need to express a heterologous SCR protein on pollen and "frequently close" its own SCR protein. The second step is to restore pollen to a "self" state. The best condition is that the heterologous SCR protein functional domain is exposed outside the cell membrane when the pollen is not pollinated, and the self SCR protein functional domain is hidden inside the cell. During pollination, the heterologous SCR protein and the self SCR protein are quickly turned over by receiving the stigma signal. However, it is very difficult to do so, because SCR is an integral membrane protein, and flipping it needs to be achieved together with membrane lipids, and the regulatory mechanism of plant membrane protein flipping is not clear, which means that we can hardly design the signal pathway to regulate flippase after pollen receives stigma signal.
The other mechanism is much clearer. We can use this mechanism to design a system to regulate pollen compatibility. The low expression of PCP-B small peptide in engineered rice pollen leads to the competitive advantage of RALP33 small peptide autocrine from stigma combined with FER/ANJ receptor kinase. The stigma is at a high level of reactive oxygen species, and the pollen can not be hydrated and germinated, thus realizing the abortion of engineered rice pollen after dispersal. Introducing another group of receptors and ligands on the membrane of pollen cells and Stigma Papilla Cells as receptors for self pollination to restore the expression level of pollen PCP-B small peptide ensures the flexible regulation of engineering rice breeding while preventing gene drift. In the selection of introduced receptors and ligands, we comprehensively considered the convenience of regulating expression and safety, selected FLS2-BAK1, a bifunctional receptor in plant immunity that can simultaneously activate calcium signaling pathway and MAPK cascade, and flg22 subunit of bacterial flagellin of strains that are safe for agricultural production and the environment, as the ligand on Stigma Papilla cells. In addition, we also plan to screen the phosphorylation sites on PCP-B by molecular dynamics and molecular docking modeling, and introduce CaM binding sites that can change the charge distribution of PCP-B.
In conclusion, we hope that the functions of this system are as follows (mechanism diagram is attached below).
For scattered pollen: PLT1/2, the key transcription factor of OsPCP-B, cannot bind to pPCP-B due to its binding to CaM. PCP-B is expressed at a low level or even not. It is absolutely inferior in the competitive binding of FER/ANJ receptor kinase in wild rice stigmas. The high level of reactive oxygen species in wild stigmas refuses to be recognized by engineering rice pollen.
For self pollinated pollen: flg22 protein on self stigma mastoid cells binds to FLS2 receptor of pollen cells, BAK1 receptor binds to FLS2 receptor and is activated by its phosphorylation, the activated BAK1 receptor recruits and allosterically activates BIK1 protein, and BIK1 protein is phosphorylated by intracellular phosphate groups to activate MAPK cascade and open calcium channels. After MAPK is activated, it enters the nucleus to phosphorylate and activate PLT1/2. After Ca2+ channel is opened, the intracellular Ca2+ concentration increases, CaM detaches from PLT1/2, pPCP-B is activated and enhanced, and PCP-B is highly expressed in pollen cells and localized to the cell membrane. PCP-B small peptide can quickly restore its advantage in the competitive binding of FER/ANJ receptor kinase in stigma, inhibit the level of reactive oxygen species in stigma, and ensure the hydration and germination of pollen.

Fig. 3 Diagram of regulation mechanism of pollen compatibility regulation system

• Laws & Regulations

According to Articles 13 and 14 of China's current effective regulations on the safety management of agricultural genetically modified organisms, during the competition, our laboratory research carried out strict operational safety management to ensure that genetically modified organisms would not be leaked or spread to the environment, and only carried out laboratory research activities, without higher security level release activities, which is legal.
According to the "guidelines for plant safety evaluation of gene editing for agriculture (Trial)" issued by the Ministry of agriculture and rural areas of China in 2022, our project uses CRSIPR/Cas9 gene editing technology to carry out site directed mutagenesis of two protein coding genes in rice, which does not involve the introduction of foreign genes, in line with the guidelines, and has carried out project safety assessment according to the guidelines.