Phytobrick collection

CRISPR technology has revolutionized biological science. Its potential and versatility are allowing scientists to carry out researches they had not ever dreamt before. Moreover, Synthetic Biology is starting to be instituted in plant science, for instance, the introduction of Phytobricks as a new iGEM standard. In this regard, we provided iGEM’s registry with Phytobricks standardized CRISPR related parts.

We provided with the necessary Phytobricks to use split Cas9 system in any desired plant, according to the phylosophy of our project. This strategy allows introducing Cas9 endonuclease through autoreplicative viral vectors of choice, which makes CRISPR/Cas9 editing more efficient and personalized. Furthermore, we contributed with tools to determine gRNA efficiency in vivo with our testing system. In this respect, we add to the registry the standard and modular parts needed to build a genetic circuit to test any possible gRNA through luciferase assays.

Essentially, we contributed with a compilation of Phytobricks aiming to make plant genome editing with CRISPR/Cas9 easier and more straightforward.

Split-Cas9 parts

The parts created to deliver Cas9 into plants are based on split-Cas9. Both parts of the split-cas9 are in pUPD2, so they can be inserted in the adapted viral vector of choice, using Phytobricks overhangs.

The inteins used to fuse the Cas9 are the inteins of 2014 Heidelberg Team (BBa_K1362400 and BBa_K1362401). We have tested these parts in plant, with success.

• BBa_K2017000. C-split Cas9 + DnaE C-intein: half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, N-split Cas9 + DnaE N-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.
• BBa_K2017001. N-split Cas9 + DnaE N-intein: half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, C-split Cas9 + DnaE C-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.

gRNA testing system parts

Our team has created a modular and standard system to test the efficiency of gRNA in vivo in plants. It includes all the parts needed to make the assembly of a testing system with the target chosen by the plant breeder.

• BBa_K2017002. 35s promoter + 5’ region: Promoter of cauliflower mosaic virus (CaMV), used for constitutive expression in plants. It is fused with the 5’ region of the N. benthamiana polyubiquitin gene, which has a high expression rate. It can enhance the expression of the coding region.

Linkers

We use firefly luciferase protein as reporter in our testing system. It oxidates its substrate luciferin, emitting light during the process. It can act as a reporter protein when used with a promoter. This part is made to be fused downstream to other coding sequence, so it includes a linker in order to let the luciferase acquire the correct tertiary structure. We tested different linkers.
The luciferase does not include ATG (Met) to initiate translation, as it needs to be fused to other coding sequence on the 5’. The translation must begin in the coding sequence fused upstream. The linker includes a random nucleotide in 5’, to change the reading frame of the luciferase. That way, it will not be translated unless an indel is produced in the coding region to which the part is fused

• BBa_K2017003. SAGTI linker + Luciferase: luciferase with the linker SAGTI.
• BBa_K2017004. RSIAT linker + Luciferase: luciferase with the linker RSIAT.
• BBa_K2017005. AEK linker + Luciferase: luciferase with the linker [A(EAAAK)<<sub>>3<<sub>1>A]
• BBa_K2017006. RSIAT+TEV linker + Luciferase: luciferase with the linker RSIAT+TEV.

Devices

We designed gRNA testing system devices to try two targets, Ga20ox of rice and TFL of Orange. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user (TFL and Ga20ox, for example), only taking into account the required overhangs (5’-AATG-3’ and 5’-TTCG-3’).
The device includes a promoter 35s, 20 nucleotides of the Ga20ox or TFL gene, the reporter luciferase with SAGTI, RSIAT, AEK or RSIAT+TEV linker and the terminator Tnos.
Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the inserted gene fragment to put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.

• BBa_K2017007. 35s:5’+ Ga20ox consense + SAGTI-Luciferase + Tnos: gRNA testing system device with the consense region of the Ga20ox gene and the SAGTI linker.
• BBa_K2017008. 35s + Ga20ox consense + RSIAT-Luciferase + Tnos: gRNA testing system device with the consense region of the Ga20ox gene and the RSIAT linker.
• BBa_K2017009. 35s + Ga20ox consense + AEK-Luciferase + Tnos: gRNA testing system device with the consense region of the Ga20ox gene and the AEK linker.
• BBa_K2017010. 35s + Ga20ox consense + RSIAT+TEV-Luciferase + Tnos: gRNA testing system device with the consense region of the Ga20ox gene and the RSIAT+TEV linker.
• BBa_K2017011. 35s:5’+ TFL consense + SAGTI-Luciferase + Tnos: gRNA testing system device with the consense region of the TFL gene and the SAGTI linker.
• BBa_K2017012. 35s + TFL consense + RSIAT-Luciferase + Tnos: gRNA testing system device with the consense region of the TFL gene and the RSIAT linker.
• BBa_K2017013. 35s + TFL consense + AEK-Luciferase + Tnos: gRNA testing system device with the consense region of the TFL gene and the AEK linker.
• BBa_K2017014. 35s + TFL consense + RSIAT+TEV-Luciferase + Tnos: gRNA testing system device with the consense region of the TFL gene and the RSIAT+TEV linker.