Optogenetic Kill Switch

Introduction

The optogenetic kill switch is the unification of two different genetic systems: pDusk, an artificially-created light-sensing promoter system; and MazEF, a toxin-antitoxin system native to E. coli.

Integral to the

The "kill" component of the kill switch, MazEF¹, is a so-called toxin-antitoxin (TA) system. As the name implies, the system consists of two components: a toxin, MazF; and its complimentary antitoxin, MazE. The MazF protein functions as an endoribonuclease, cleaving mRNA and thereby inhibiting transcription, which is ultimately lethal for the cell. MazE forms a complex with MazF, preventing it from functioning. However, MazE is a more labile protein than MazF, degrading faster. For this reason, TA systems like MazEF are also known as addiction modules; the organism expressing the system becomes "addicted" to the antitoxin and is negatively affected should expression stop. In its native organisms, MazEF has a regulatory function. Under ordinary conditions, the proteins are co-expressed and the organism stays stable. However, under stressful conditions, such as nutrient starvation, this expression ceases. MazF is then free to cleave essential mRNAs, causing what is inferred to be a state of bacteriostasis.² This process is reversible up until approximately 6 hours after taking effect, the so-called Point of No Return. Much like programmed cell death (PCD) in multicellular organisms, this prevents excess growth the bacterial population cannot afford, improving viability.

The optogenetic kill switch is designed to exploit this mechanism. Rather than remaining expressed until the population encounters stress, MazE is expressed under pDusk. MazF, on the other hand, is expressed constituitively. This means that, in the darkness of the beehive, both toxin and antitoxin are formed, allowing the cell to remain stable. However, when the cell is exposed to light over a longer period of time, antitoxin expression under pDusk drops and the equilibrium shifts in favor of the toxin. Thus, escaped bacteria die.

Construction of genetic circuit

Both MazE and MazF have been registered as BioBricks. However, as of yet, neither are available. For this reason, we chose to isolate both genes directly from E. coli genomic DNA.

Cas9-based killswitch

To further minimize the risk of BeeT escaping the beehive, we aimed to include another safety mechanism in parallel with the light kill switch. An idea that came up was using a bacterial strain developed by Mandell and colleagues (2014)¹ as a chassis for BeeT. This “biocontainment strain” can be confined to a certain area because it is auxotrophic for a synthetic amino acid, para-L-biphenylalanine (BipA). Several essential proteins of this bacterial strain were engineered to function only when BipA is incorporated in the active site, leading to death of the bacterium when the synthetic amino acid is not available. In our case BipA should be applied to the beehive, so it cannot survive either when it escapes in the environment, or when the beekeeper ceases to supply BipA.

A drawback of this system is that, even though the biocontained organism dies as soon as it is deprived of the synthetic amino acid, its heterologous DNA might remain in the environment. Since DNA is rather stable given the right circumstances², there is a risk it is be taken up by other bacteria through horizontal gene transfer^3,4. In our project, we sought to complement the biocontainment strain by creating a switch to destroy heterologous DNA, depending on the presence of BipA. Additionally, the switch can be used to further strengthen the auxotrophy for the SAA by targeting genomic DNA as well.

Using Cas9 to reinforce auxotrophy for a synthetic amino acid

To this end, we aimed to engineer Streptococcus pyogenes Cas9 to switch from a catalytically dead form (dCas9)⁵ to a partially active nickase form (nCas9)⁶ as soon as BeeT leave the hive. nCas9 is a Cas9 version that nicks DNA rather than making double strand breaks. However, it was shown that nCas9 is able to cause double strand breaks when two spacers are available to nick DNA on opposite strands in close proximity.

The switching between dCas9 and nCas9 depends on whether a BipA is incorporated into Cas9 (figure 1). Two residues known to be essential for DNA cleaving are replaced with BipA: Asp10 and His840. Additionally, one of these residues is changed to alanine, as changing only one of these essential residues will make a switch between fully active Cas9 and nCas9. To incorporate the BipA, the codon for the native AA is replaced by the TAG stop codon. To translate the TAG codon, a tRNA that recognizes the TAG codon (tRNA_CUA) and an amino-acyl-RNA-synthetase (aaRS) that charges the tRNA with BipA are provided⁷. A genetically recoded strain that lacks any TAG stop codons as well as release factor 1⁸ is used to make translation of the TAG codon more efficient.

BipA will be loaded on the tRNA_CUA as long as the E. coli stays in the hive, where BipA is supplied. This results in the formation of dCas9. When E. coli escapes from the hive, no charged tRNA_CUAs are available, the Cas9 transcript cannot be translated and dCas9 is not formed.

To switch from expression of dCas9 to nCas9, dCas9 is used to repress transcription^9,10 of another aaRS and tRNA_CUA that is charged with a natural amino acid, for example aspartate¹¹. When the organism is deprived of BipA, the repression is released. With the tRNA_CUA(Asp) available, the Cas9 transcript can be translated again. However, this time Aspartate is incorporated in Cas9 instead of BipA, resulting in the formation of nCas9 rather than dCas9. The nCas9 is able to cut any DNA for which two suitable spacers are offered, including all heterologous DNA.

Cloning and expression of Cas9 variants

Ala10TAG and Ala840TAG mutations were introduced separately in dCas9 (Addgene plasmid # 46569)¹⁰. A vector to translate the TAG stopcodon to incorporate BipA was already available from the biocontainment strain¹ (pEVOL-BipA). pEVOL-Asp was constructed to translate the TAG stopcodon to incorporate Aspartate instead.

To test translation of the TAG stopcodon and incorporation of BipA in Cas9, the Expresso system for rhamnose induced expression from Lucigen was used¹². Bacterial cultures transformed with Cas9-expresso constructs, and in case of a mutant Cas9, either with pEVOL-BipA or pEVOL-Asp were used for extraction of Cas9 variants. SDS-PAGE of the FPLC purified samples showed clear expression of both Cas9 and dCas9, and lower expression of dCas9-Ala10BipA. A faint band could be seen for dCas9-Ala10Asp, but since a control sample without any synthetic amino acid also displayed a (very) faint band, this result is not conclusive (figure 2).

In vitro Cas9 cleaving assays

To assess the functionality of our Cas9 variants, in vitro Cas9 cleavage assays were performed. An RFP PCR product of 4140 bp was chosen as the target for cleaving to generate two fragments: one ~3100 bp fragment and one ~1040 bp fragment. All targets were chosen in close proximity; meaning all cleavage products would be roughly the same length, and targets on opposite DNA strands could generate a double strand break when incubated with dCas9-Ala10Asp (nCas9).

From the assays, it can be concluded that our purified Cas9 is functional using several gRNAs targeting a different part of RFP, as the linear target was in all cases cleaved at least to some extend. As expected, dCas9 does not have this effect (one more assay, and I can have an indication that ala10BipA is also inactive). When two gRNAs that target the RFP gene on opposite strands are offered to dCas9-Ala10Asp, it is expected to see some cleaving caused by the two nicks in close proximity. Indeed, some cleaving was observed. However, also some cleaving was observed when offering only one gRNA. It has been shown that the His840Ala version of nCas9¹³ (which is basically the same as our dCas9-Ala10Asp) has some double stranded DNAse activity. Further testing is needed whether this caused the cleaving by dCas9-Ala10Asp in our case, or that it is an artefact of some sort (figure 3).

In conclusion: in this part of our project we showed that an artificial amino acid can be incorporated in dCas9 in response to the TAG stopcodon (and that this version remains catalytically inactive → remains to be seen). We also tried to incorporate Aspartate in response to the TAG stopcodon, and while we have some indications that this restored nickase activity, further testing is needed to verify whether this is the case.

References

 

References for optogenetic kill switch

1. Engelberg-Kulka, H., Hazan, R., Amitai, S. (2005). mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. Journal of Cell Science 118, 4327-4332. ↩

2. Amitai, S., Yassin Y., Engelberg-Kulka, H. (2004) MazF-Mediated Cell Death in Escherichia coli: a Point of No Return. Journal of Bacteriology vol. 186 no. 24 8295-8300. ↩

References for Cas9-based kill switch

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2. Romanowski, G., Lorenz, M. G., Sayler, G., & Wackernagel, W. (1992). Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Applied and Environmental Microbiology,58(9), 3012-3019. ↩

3. Thomas, C. M., & Nielsen, K. M. (2005). Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nature reviews microbiology, 3(9), 711-721. ↩

4. Smillie, C. S., Smith, M. B., Friedman, J., Cordero, O. X., David, L. A., & Alm, E. J. (2011). Ecology drives a global network of gene exchange connecting the human microbiome. Nature, 480(7376), 241-244. ↩

5. Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., ... & Church, G. M. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology, 31(9), 833-838. ↩

6. Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., ... & Skarnes, W. C. (2014). Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods, 11(4), 399-402. ↩

7. Xie, J., Liu, W., & Schultz, P. G. (2007). A genetically encoded bidentate, Metal‐Binding amino acid. Angewandte Chemie, 119(48), 9399-9402. ↩

8. Lajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H. R., Haimovich, A. D., Kuznetsov, G., ... & Rohland, N. (2013). Genomically recoded organisms expand biological functions. Science, 342(6156), 357-360. ↩

9. Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183. ↩

10. Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research, 41(15), 7429-7437. ↩

11. Pastrnak, M., Magliery, T. J., & Schultz, P. G. (2000). A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair for evolving an organism with an expanded genetic code. Helvetica Chimica Acta, 83(9), 2277-2286. ↩

12. Expresso® Rhamnose Cloning & Protein Expression System ↩

13. Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., ... & Skarnes, W. C. (2014). Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods, 11(4), 399-402.↩