Team:Valencia UPV/Proof

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gRNA Testing System

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• First Stuff

Title

gRNA Testing System

Abstract

We have designed a modular gRNA testing system in order to check if the gRNA provided by the Data Processing Software works as expected on the plant variety to improve. This system works as a genetic switch that remains OFF if the gRNA does not work, and turns ON when the Cas9 does a double stranded break in the target. The device performance is based on the fact that the luciferase reporter gene is out of its reading frame. When CRISPR/Cas9 system works as expected, meaning that the gRNA is well designed, it will introduce indels in our device and the luciferase will be placed on its correct reading frame. Therefore, we will be able to detect bioluminescence with a luciferase assay.

gRNA Testing System

gRNA is a key element in genome editing with CRISPR/Cas9 system, since it leads endonuclease Cas9 where the modification must be produced. That means gRNA must work properly in the plant variety that we want to improve.

The plant breeder could use the gRNA provided by our software directly in his plant. However, lots of plant species take a long time to grow. For example, orange trees need at least 1-2 years to exceed the first growth stage, and 3-4 years to produce the first fruits. If the gRNA didn’t work properly, the plant breeder would had lost a valuable time waiting to see a phenotypic improvement on his plant. It would take a long time just to know if the gRNA has worked or not. Due to that, it’s necessary to previously check the gRNA proper functioning.

Why would not our gRNA work?

Our open-source database includes genes of interest, which can be selected by plant breeders according to their needs. It also directly provides the optimal gRNA which, afterwards, must be ordered to synthesize. The gRNA is obtained from gene consensus sequences acquired from big databases such as NCBI or Sol Genomic. However, the consensus sequence may match or not with the specific variety to improve. Insertions and deletions - Indels - and Single Nucleotide Polymorphisms - SNPs - occur spontaneously and randomly in the genome of different plant varieties. That means the gRNA obtained by our data processing software could not work optimally on the desired variety. This makes even more necessary to test the gRNA before using it in the specific variety. Furthermore, even if the gRNA matches perfectly with the target sequence, mutagenesis may not occur - or be less efficient - due to gRNA secondary structure problems.

gRNA testing methods

The current methods used to test the efficiency of CRISPR/Cas9 -and therefore that can be used to test the gRNA- are digestion with restriction enzymes or digestion with T7 endonuclease and subsequent sequencing. However, these methods are not suitable if we want to make accessible and easy the testing of the gRNA.

• Digestion with restriction enzymes: when choosing the target within the gene, it is mandatory to choose a target including a restriction site where the Cas9 will make the DSB. Therefore, in order to check if the Cas9 produced the mutation, a digestion is performed with the restriction enzyme, and an electrophoresis gel is carried out. If the Cas9 cuts, the restriction site will be lost and the enzyme will not cut. Therefore, in the electrophoresis gel, a band with higher molecular weight should be observed. If mutation did not occur, two bands should be observed, since the enzyme can recognize the site and cut.
• Problem: the range of possible targets is reduced, because they must contain a restriction site exactly in the place where the Cas9 cuts. Additionally, when you find a target with a restriction site you might not have the needed enzyme. Buying it may imply a high cost, not affordable for everyone.

• Digestion with T7 endonuclease: this strategy is similar to the restriction enzymes one, yet it uses the T7 endonuclease. This endonuclease cuts where it finds heterodimers. When Cas9 cuts, due to the non-homologous end joining DNA repair mechanism - NHEJ -, plant cells introduce indels. After a PCR amplification of the region, heterodimers can be obtained from a denaturation and reannealing step. When they are annealed, they might bind with a strand which is not exactly complementary, producing heterodimers that T7 can cut. Therefore, in an electrophoresis gel, a high molecular weight band will be observed if there is not a cut, while, two shorter bands will appear if T7 endonuclease cuts and so, Cas9 is well working.
• Problem: the T7 endonuclease is outrageously expensive. Buying it may imply even a higher cost, not affordable for everyone.

In order to provide an efficient solution to the drawbacks explained above, we have engineered a gRNA Testing System. In this strategy, we use Nicotiana benthamiana due to its fast growth and all the benefits provided by a model plant. Our methodology is based in the GoldenBraid Assembly System, that allows us to get a modular and standard system, two of the mainstays of Synthetic Biology field.

Our device

The fragment of genome that we are going to target in our plant is inserted in the Testing System following the concept of modularity. Based on Agrobacterium tumefaciens infection and using a reporter gene in our device, we will be able to know how efficient the provided/designed gRNA is.

First of all, plant breeders have to carry out a genomic DNA extraction of the plant they want to modify. Next, they need to amplify the region they are going to target. The targets needed to this amplification are provided by our Data Processing Software. The amplified target sequence is introduced in N. benthamiana as part of the device with the luciferase reporter.

This device is introduced in the plant along with the corresponding gRNA and Cas9 construction. If the gRNA works on the desired variety, we will be able to detect it easily with a luciferase assay.

Our system is designed to allow the detection of luminescence. Thus, a luciferase assay is used to study gene expression rates, since it is fast and the analysis of each sample only requires a few minutes. Moreover, it is extremely sensitive and the results are very accurate, allowing us to obtain quantitative results. Originally the system is OFF since luciferase genetic sequence is not in the correct frame. When Cas9 cuts, indels appear, system turns ON, so luciferase gene is in the correct frame and it will be correctly translated. In that case, the plant breeder could check the genome editing in a simple way. Cells are assayed for the presence of the reporter by directly measuring the enzymatic activity of the reporter protein on luciferin substrate.

Parts of the device

P35s : 5’ region : TARGET : Linker (+2) : LUC : Tnos

• P35s: It is a strong constitutive promoter derived from cauliflower mosaic virus (CaMV). It is widely used in plants to improve the level of the expression of foreign genes effectively in all tissues.

• 5’ region: 5’ region from the N. benthamiana polyubiquitin sequence (Accession number: Nbv5.1tr6241949) is used due to its high expression rate in every plant tissue. It can help our gene to express itself and ensures that the construction is expressed. It contains BsmbI and BsaI recognition sites with AATG in 3’ as overhang that allows us to ligate with the amplified target.

• Target: Plant breeders will obtain the selected gene from their original plant by PCR amplification with the primers provided by our Data Processing Software. These primers are designed in order to obtain amplicons with the overhangs needed to insert the target in our device, corresponding to GoldenBraid grammar (prefix: AATG, suffix: TTCG). It’s mandatory to make sure that this region doesn’t contain any stop codon in frame +1, but neither in frame +2, so when an indel happens the reading frame will not be disrupted. The software finds the optimal target with its corresponding gRNA.

• Linker SAGTI (Ser-Ala-Gly-Thr-Ile): it is a flexible peptide linker between the target and the luciferase, so it allows the luciferase to acquire the correct structure, avoiding interaction with the target. Thus, luciferase assay will be carried out in a successfully way. As it can be seen in the figure 1, the first part of the device is in ORF +1 whereas the luciferase is in ORF +2. The device is designed so that there is an extra nucleotide before the linker sequence to change the reading frame. If this nucleotide were after the linker sequence, when Cas9 cut, the reading frame of the linker would change, and the amino acids translated would not be the correct ones. Figure 1 shows how it will be translated after the indels.

Figure 1. Device after endonuclease Cas9 cut and indels production. a) The extra nucleotide is located before the linker, so when indels change the frame, the linker and the luciferase are in the correct ORF. b) The extra nucleotide is located after the linker. Thus linker’s frame will be different from luciferase’s one after indel occurs.

Luciferase: It is widely used as a reporter enzyme. At the beginning, luciferase gene is out of the reading frame due to the presence of an extra nucleotide. After CRISPR/Cas9 acts indels occur. These insertions and deletions of nucleotides produce a frameshift and change luciferase’s frame in the right frame +1, so it will be correctly translated. In the experimental design, the first methionine has been removed just to prevent the unintended translation of the luciferase. Therefore appearance of false positives will be avoided.

• Tnos: Nopaline synthase terminator of the nopaline synthase gene of Agrobacterium tumefaciens. It is used for gene transfection.

The plant breeder will be provided with a pUPD2 vector with P35s:5’region, another pUPD2 with linker:luciferase, and a pUPD2 ready to insert the target directly obtained from their plant variety. Using GoldenBraid assembly, the breeder will insert the target within the pUPD2. Afterwards, in a GoldenGate ligation reaction, all the parts of our device can be assembled, obtaining the complete gRNA testing system construction. Afterwards A. tumefaciens will be transformed with the obtained plasmid and it will be used to agroinfiltrate N. benthamiana leaves. Four days post infiltration, the breeder will be able to perform the luciferase assay.

Bibliography

• Biolabs, N.(2016).Measuring Targeting Efficiency with the T7 Endonuclease I Assay | NEB. (online) Neb.com. Available at: https://www.neb.com/applications/cloning-and-synthetic-biology/genome-editing/measuring-targeting-efficiency-with-the-t7-endonuclease-i-assay (Accessed 27 Jul. 2016).

• Pauli, S., Rothnie, H., Chen, G., He, X. and Hohn, T. (2004). The Cauliflower Mosaic Virus 35S Promoter Extends into the Transcribed Region. Journal of Virology, 78(22), pp.12120-12128.

• Guilley, H., Dudley, R., Jonard, G., Balàzs, E. and Richards, K. (1982). Transcription of cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell, 30(3), pp.763-773.

• Sefapps02.qut.edu.au. (2016). Benthamiana Atlas. (online) Available at: http://sefapps02.qut.edu.au/atlas/tREXXX2new.php?TrID=Nbv5.1tr6241949 (Accessed 27 Jul. 2016).

• Chen, X., Zaro, J. and Shen, W. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), pp.1357-1369.

• Holden, M., Levine, M., Scholdberg, T., Haynes, R. and Jenkins, G. (2009). The use of 35S and Tnos expression elements in the measurement of genetically engineered plant materials. Anal Bioanal Chem, 396(6), pp.2175-2187.

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