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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.

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

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.

Figur:

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).