Team:Valencia UPV/Hardware/Reagents

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• MODEL
  • OVERVIEW
  • Cas9:gRNA Complex Formation
  • R-Loop Formation
  • Cleavage and Reading Frame Shift
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Reagents

Index

• Split-Cas9
• gRNA Testing System
• Why wouldn’t our gRNA work?
• gRNA testing methods
• Our device
• Parts of the device (gRNA Testing System 1.0)
• New parts of the device (gRNA Testing System 2.0)
• References:

Split-Cas9

We provide all the necessary constructions in order to use autoreplicative vectors as Cas9 delivery system. That means, providing both Cas9 halves fused with its corresponding intein inserted into two different vectors Tobacco Mosaic Virus (TMV) and Potato Virus X (PVX).

gRNA Testing System

Standardization and modularity are two of the mainstays of Synthetic Biology. Bearing that in mind we have design, built and tested a Phytobricks standardized genetic circuit that allows to analyze the efficiency of a given guide RNA (gRNA) in vivo.
We have designed a modular gRNA testing system that allow us to determine the efficiency of any provided gRNA in vivo. This system works as a genetic switch that remains OFF until Cas9 endonuclease, guided by a gRNA, performs a double stranded break inside the target sequence. The device performance is based on the presence of a luciferase reporter gene out of frame. When a double strand break (DSB) occur due to Cas9 activity, a frameshift mutation may appear. Thus, luciferase’s correct reading frame will be recovered and bioluminescence will be detected through a luciferase assay.
gRNA is a key element of CRISPR/Cas9 system. It leads Cas9 endonuclease to the selected target in order to produce a mutation. In order to maximize the mutation efficiency in the desired crop variety gRNA must work properly. Therefore gRNA design must be optimal.
Plant breeders could use the gRNA provided by our software or any other one to directly edit their desired crop. However, this process usually takes long time and work turns time-consuming and in some cases effortless. It would take a long time just to know if the gRNA has worked or not. For instance, orange trees need at least 1-2 years to exceed the first growth stage, and 3-4 years to produce the first fruits. Therefore, we encourage plant breeders to use our gRNA testing system in order to determine the efficiency of their RNA.

Why wouldn’t 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 Genomics. 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 plants in nature. That means the gRNA obtained by our data processing software could not work optimally on the desired variety. This makes almos mandatory to test the gRNA in vivo 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, as described by Summer et al. (1).

gRNA testing methods

The current methods used to test the efficiency of CRISPR/Cas9 consists in digesting amplified mutated region with restriction enzymes or with T7 endonuclease. However, these methods are not suitable if we want to be able to test any gRNA with a reproducible method. Therefore, a new modular and standard gRNA in vivo testing method claims to be developed.

• Digestion with restriction enzymes: the major drawback of this method relies on the fact that only targets that includes a restriction site in DSB region are eligible. In order to check if the mutation has occured, a digestion is performed with the chosen restriction enzyme, and an electrophoresis gel is carried out. If mutation occured, the restriction site will be lost and the enzyme won’t 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. Moreover, determining efficiency ratios through this method is difficult and not very accurate since it is based on band signal intensity comparison.

• 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 (2). 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 recognize and 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. Moreover, this method does not allow to analyze the obtained mutation by sequencing since those frgaments with the mutation will be cut.

In order to provide an efficient solution to the aforementioned drawbacks, we have engineered a gRNA Testing System. In this strategy, we chose Nicotiana benthamiana as chassis due to its fast growth and all the benefits provided by a model plant. Our methodology is Phytobricks standardized, thus turning this system into a modular and standard one, two of the mainstays of Synthetic Biology.

Our device

Any selected target can be inserted in the Testing System since its Phytobricks standardized and its overhang sequences are well defined. Based on Agrobacterium tumefaciens infection and using luciferase as reporter gene, we will be able to know how efficient the provided/designed gRNA is.
First of all, plant breeders must perform a genomic DNA extraction of the plant they want to modify. Next, they need to amplify the region they are going to target. The primers needed to carry out this amplification are provided by our Data Processing Software. The sequence of the amplified target is then analyzed by sequencing. Next step consists in ordering overlapping oligonucleotides corresponding with the analyzed target sequence. Finally the target is introduced in the construction of our testing system, introduced in A.tumefasciens and next in plant leaf cells by agroinfiltration (Fig. 1).

(Fig. 1) Schematic of the insertion of the target inside the construction of gRNA Testing System circuit.

This device is introduced in the plant along with the corresponding gRNA and Cas9 construction. If the gRNA leads Cas9 to the target and mutations are produced, bioluminescence signal will be detected through a luciferase assay.
The main advantages of this kind of assays are its straightforward protocol (the analysis of each sample only requires a few minutes) its enormous sensitivity and its accuracy on the obtained results. Thus, allowing us to obtain quantitative results.
The system is designed in order to be in OFF state until the DSB and subsequent repair is produced. When Cas9 cuts, indels appear, luciferase open reading frame returns to the correct one and system turns ON (Fig. 2 and 3). Leaf samples are assayed for the presence of the reporter gene by directly measuring the enzymatic activity of luciferase enzyme in the presence of known concentration of luciferin.

(Fig. 2) Schematic of the function of the genetic circuit that composes the gRNA Testing System. Only when DSB and subsequent repair is produced the luciferase’s open reading frame will shift to the correct one (ON state).

(Fig. 3) 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.

Parts of the device (gRNA Testing System 1.0)

All the parts used in the construction of the gRNA testing system 1.0 and following versions are described here.

• 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 (3).
• 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 (4). It can help our gene to express itself and ensures that the construction is well 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 Phytobricks 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 won’t 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. 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 Figure 3, 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 that changes the reading frame. If this nucleotide were after the linker sequence, when Cas9 cut, the reading frame of the linker will change, and the amino acids translated won’t be the correct ones. Figure 3 shows how it will be translated after the indels.
• 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 (5).

New parts of the device (gRNA Testing System 2.0)

After building the device different luciferase were performed in order to test its feasibility. Once the obtained results were analyzed (See results here), we determined that the system were working well but its performance could be improved. Bearing that in mind, we resolved to remove the 5’ region in order to obtain higher levels of luciferase expression and also to test different linkers and choose the most appropriate one. The new selected linkers are:

• EAAAK linker: rigid helical linker with the sequence of A(EAAAK)nA (n =3) that effectively separates protein domains as reported by Chen et al. (6). An extra nucleotide was added following the same strategy design that with SAGTI linker.
• RSIAT linker: short linker that separates protein domains and that have been succesfully used BiFC experiments (7). An extra nucleotide was added following the same strategy design that the aforementioned linkers.
• RSIAT+TEV linker: RSIAT linker (7) followed by a TEV protease recognition site. Therefore Tobacco Etch Virus NIa protease will recognize the sequence and cleave it, obtaining two different peptides as result. On one side target plus linker peptide, and on the other side luciferase protein. With this linker we aim to obtain a more orthogonal design in which the target sequence has minimal interference with luciferase protein. Thus, luminescence signal will not depend on the target.

References:

1. Thyme S, Akhmetova L, Montague T, Valen E, Schier A. Internal guide RNA interactions interfere with Cas9-mediated cleavage. Nature Communications. 2016;7:11750.
2. Biolabs N. Determining Genome Targeting Efficiency using T7 Endonuclease I | NEB (Internet). Neb.com. 2016 (cited 27 July 2016). Available from: https://www.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i
3. Pauli S, Rothnie H, Chen G, He X, Hohn T. The Cauliflower Mosaic Virus 35S Promoter Extends into the Transcribed Region. Journal of Virology. 2004;78(22):12120-12128.
4. Benthamiana Atlas (Internet). Sefapps02.qut.edu.au. 2016 (cited 27 June 2016). Available from: http://sefapps02.qut.edu.au/atlas/tREXXX2new.php?TrID=Nbv5.1tr6241949
5. Holden M, Levine M, Scholdberg T, Haynes R, Jenkins G. The use of 35S and Tnos expression elements in the measurement of genetically engineered plant materials. Analytical and Bioanalytical Chemistry. 2009;396(6):2175-2187.
6. Chen X, Zaro J, Shen W. Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews. 2013;65(10):1357-1369.
7. Hu CKerppola T. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nature Biotechnology. 2003;21(5):539-545.

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