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CRISPR all in one

Most CRISPR-technology vectors with both gRNA and Cas9 expression has one set of regulatory mechanism for each of the coding sequences. Due to CAS9s immense size there is often problems working with this technology and having two regulatory sequences does not help. This effect is especially detrimental while using complex eukaryotic regulatory sequences like the 1077 bP long promotor pCLN1, used in iGEM 2014 Team Gothenburg.

To minimize the need for regulatory sequence we introduced a new system which has a similar effect as a bacterial operon. The system is capable of regulating one protein and one RNA-sequence in eukaryotes using only one promotor. We call it CGC1 (Cas9 gRNA complex one) and used in the signal amplification mechanism in our detection system.

¨To get both of the complex components a set or requirements must be set in the RNA stage of the central dogma molecular biology. First, the coding sequence for CAS9 be flanked by the ‘5 cap and the poly A- tail to be transported outside the nuclear envelope and be translated into the protein. Second, the gRNA must be free from polyA tail and 5’ cap stop relocation into the cytoplasm and degradation

To get both of the complex components a set or requirements must be set in the RNA stage of the central dogma molecular biology. First, the coding sequence for CAS9 be flanked by the ‘5 cap and the poly A- tail to be transported outside the nuclear envelope and be translated into the protein. Second, the gRNA must be free from polyA tail and 5’ cap stop relocation into the cytoplasm and degradation

The solution to the problem uses two simple parts, hammer head ribozymes and synthetic poly-A tails. The hammer head ribozymes are functional secondary RNA structure which induces its self-cleavage. The use of ribozymes to create gRNA are quite common in the research community but also in iGEM, an example of a team using ribozymes to produce gRNA in vivo are iGEM 2014 Team Gothenburg. The other CGC1 part the synthetic poly A is used as the new poly-A tail after ribozyme cleavage ensuring the cas9 cytoplasm translocation and translation.

Figure1, The sequence for the hammer head ribozyme.

https://2015.igem.org/File:ChalmersgothenburgOtherApplications2.png

Figure 2,The Sequence of the Synthetic Poly-A tail

https://2015.igem.org/File:ChalmersgothenburgOtherApplications1.png

To use the two prats in the system, the proteins sequence should be put the closet to the promotor and RBS. The protein sequence should be followed by the synthetic poly-A tail and then a hammer head ribozyme. After the ribozyme the wanted free RNA sequence should be placed and lastly another ribozyme.

Figure:

https://2015.igem.org/File:ChalmersgothenburgOtherApplications3.png

Figure 3, Mechanism of the CGC1 system. The CGC1 gene is transcribed and the natural 5’ Cap and the Poly-A tail are added to the sequence. The two hammerhead ribozymes (HHR) induces its own cleavage, which separates the protein coding sequence from the gRNA. The natural 5´Cap and the synthetic poly-A tail (Poly A) ensures the translation of the protein coding sequence (Cas9).

Gibson 2.0

The world of synthetic biology is growing tremendously fast and part of it is the development of the tools used is a big part of it. Year 2003 was the BioBrick standard introduced as an intelligent way of using restriction sites to construct genetic constructions, however the methodology is limited due to the nature restriction, the DNA sequence needs to contain those specific restriction sites in the end of the fragments, and it can’t have any of those restriction site inside the DNA fragment.

2009 a methodology introduced which avoids potential problems with retraction-sites and –enzymes, Gibson assembly. The method utilizes sequence overlaps between the two DNA fragments to fuse them together. Unlike BioBrick assembly there is no requirements on the overlapping region which is one of the strengths of Gibson assembly

Figure 4, https://2015.igem.org/File:ChalmersgothenburgOtherApplications4.png

Figure 4.The mechanism of Gibson assembly. 1) An exonuclease chews of one of the two strands of the two DNA fragments (blue/green and orange/green). 2) The two fragments bind together due to their overlapping region (green). 3) A DNA polymerase fills in the gaps and a ligase repair the backbone nicks. 4) The DNA fragments are fused.

Now we introduce a concept for next generation of DNA assembly, Gibson 2.0. The concept is very similar to the normal Gibson assembly and is also compatible with normal Gibson reactions at the same time, but it removes the need for a specific overlap all together. The method is inspired by our repair mechanism and uses the normal Gibson proteins, but also a Recombinase and an oligonucleotide. The reason why no sequence overlap before the reaction isn’t needed is the exploitation of the recombinase and oligonucleotide to create an overlap during the reaction.

Figure 5, https://2015.igem.org/File:ChalmersgothenburgOtherApplications5.png

Figure 5, Mechanism for Gibson 2.0. 1) An exonuclease chews of one of the two complementary DNA strands on both of the DNA fragments. 2) One of the two DNA fragments is extended using the oligonucleotide as a reading frame. 3) The two fragments bind together due to their overlapping region 4) A DNA polymerase fills in the gaps and a ligase repair the backbone nicks. 5) The DNA fragments are fused.

Next generation marker

There is a wide range of laboratory markers available for use. One if not the most common marker resistance versus a specific chemical like antibiotic resistance. These markers are often very effective since the selective medium does not only kill your cells without the wanted DNA constructions, but it also kills of potential contaminants. However the use of antibiotics could potentially cause selective pressure for antibiotics resistance in pathogens if the antibiotics isn’t taken care of properly. If toxic chemicals are used, it could potential harm humans. Therefore extra safety measures need to be taken to combat these risks.

An alternative method would be to auxotrophic markers which is based on knocking out a protein in a pathway which synthesizes an essential biomolecule like a nucleotide or an amino acid. If the cells are grown on a normal nutrient media e.g. LB for E. coli or YPD for S. cerevisiae, the cells will survive. But if they are grown in a media without that specific biomolecule the only survivors will be the cells with the knocked out gene reinserted. The problem with auxotrophic markers could be mixing or contamination by another strain without that specific knockout.

Our concept for the next generation marker involves our repair system. The used strain should have the needed parts for the repair system but one. During transformation/transfection the last repair protein should be added and then selection is done by irradiating the sample. The wanted cells would survive while the normal cells would die, the same applies for eventual contaminants. So far it has the same effect as the chemical markers but without the downsides of auxotrophic markers. However, since the irradiation causes no chemical waste handling it would be the more ethical choice.

Space applications

The next frontier, the border to the unexplored, space. Having sustainable production of recourses is one of the big problems needing a solution before we start colonizing other planets. Mars is a possible candidate for colonization but it wouldn’t be easy. There is only a low abundance of oxygen and therefore ozone in the atmosphere. Without ozone, a lethal amount of UV radiation bombards the surface of Mars making most life from earth unable to survive. Another problem to life forms such us ours, is the lack of oxygen which leads to death by suffocation.

A solution for the colonizing would be to grow plats on mars, and when you have adult plats, then, it is only a matter of placing them in a synthetic environment like a dome. Humans could then possible live inside the dome.

All of this start with is enabled with a good mechanism for plants to survive in high levels of UV. Some projects like our neighbor team UNIK Copenhagen (https://2015.igem.org/Team:UNIK_Copenhagen) are trying to design vegetation which survives on mars. They take a lot more survival factors than UV irradiation into an account, but our repair system could be one of the pieces needed for the final product.