Improve Safety And Specificity Of Genome Editing Using Unnatural Amino Acids

CRISPR/Cas(clustered regularly interspaced palindromic repeats/CRISPRassociated) systems are bacterial and archaeal acquired immunity system that defence against reinvading foreign nucleic acids derived from bacteriophages, covering two subsystems :RNA system and Cas system. Currently, the widely applied type II spCas9 use an array of small CRISPR RNAs(crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate DNA target degradation. After DNA double strand breaking, turning on of DNA damage repair pathway such as nonhomologous end joining(NHEJ) and homologous recombination(HR), can generate insertions or deletions leading to frame-shift mutation in the exon.Compared ZFNs and TALENs, with advantages such as simple and low costs, CRISPR/Cas9 system quickly become a mainstream technology and a necessary research tool in genome editing and modern life science. But this simple future of CRISPR/Cas9 also lead to a potential bio-safe difficulty, which with the off-target effects that are persistently reported, greatly limits its further application.After decades of development, the improved site-specific unnatural amino acid technology has become a good strategy that can be implied in biocontainment of genetically modified organisms, however, there are no more reports other than George M. Church and Farren J. Isaacs. And what the point they focus on is the prokaryote only, not about the eukaryote.Here we redesign site-specific incorporation of unnatural amino acids technology to a biocontainment system of genetically modified eukaryotic multicellular. We insert a stop codon in Cas9 gene, so its existing form is not in translational but in transcriptional, if unnatural amino acid is not add to cell culture. Besides, this new CRISPR/Cas9 system offers an additional strategy in taming off-target mutagenesis.The main results and conclusions in this paper as follows:1. Constructed a method for genetically encoding unnatural amino acids in living cellsTaking bacteria, yeast, mammalian cells, and fruit fly for example, we made such results:(1) With an unnatural amino acid[ H-Lys(Boc)-OH, BocK ] and PylRS/tRNATAG pairs, we successfully created a DsRed-TAG-EGFP double fluorescence reporter system. And only BocK in the cell culture, yellow light could be captured by fluorescence microscope. Western Blot also confirmed the large fused protein existing only when BocK in. Those results revealed that unnatural amino acids can be involved in target protein through TAG, so TAG not just a termination codon, but a coding codon.(2) Based on result(1), we successfully made a CRISPR/Cas9* system, and Western Blot showed that a Cas9*(TAG-Cas9) was translated into protein when TAG as a coding codon.2. Raised and proved that site-specific incorporation of unnatural amino acids can guarantee bio-safe in CRISPR/Cas9* mediated genome editingAs the earlier model organisms, bacteria currently has been widely used in the biopharmaceuticals, biological energy and biological degradation, however, survival of microorganisms into natural environments remains a defining challenge. Recently sitespecific incorporation of unnatural amino acids is becoming a tool to guarantee bio-safe in transgene field, therefore, we hypothesize that it can also be apply in CRISPR/Cas9.The activities detection by SSA revealed that unnatural amino acid indeed was the key factor to unlock Cas9* expression, subsequently, Western Blot and RT-PCR assay demonstrated that Cas9* was not translational, but transcriptional. So it can be seen from above unnatural amino acid can be used as a molecular switch to control Cas9* expression, and which make an increasingly improvement of bio-safe in genome editing.3. Using the unnatural amino acid can improve the specificity of CRISPR/Cas9*For the CRISPR/Cas9 system, the uncured off-target effects are main limiting factors in therapeutic and molecular breeding, and the latest year various strategies have been described to reduce genome-wide off-target mutations, however, these approaches are only partially effective. In this part, we focus on the off-target mutations of CRISPR/Cas9.Firstly according to the DNA sequence in the three sites—VEGFA, EMX1 and 293-4, revealed an increased off-target with the time accumulation. And sequencing in 293-4 site and its potential two off-target sites uncovered that incorporation of unnatural amino acids during 0-12 hours dramatically eliminate the off-target effect in its two potential off-target sites, and incorporation during 19-30 hours can reach off-target to a no detect level.Above all, we firstly apply the expanding genetic code system to the CRISPR/Cas9 system to site-specifically incorporate an unnatural amino acid into the Cas9 protein, so unnatural amino acids are able to be used as a molecular switch to control Cas9 * expression, and this make an increasingly improvement of bio-safe in genome editing. In addition, this switch can reach off-target to a lower level than wild Cas9. The establishment of CRISPR/Cas9* system can expand its basic role in the applications of genome editing. I believe that the combination with other reported method that can enhance the specificity of the CRISPR/Cas9 system, will put it as a safe, accurate, and efficient tool.