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Team:Tuebingen/Killswitch

GLP.exe - Kill Switch

Kill Switch

The CRISPR/Cas3 System

Similar to the type II CRISPR/Cas9 system, the type I CRISPR/Cas3 system is part of the microbial adaptive immune system. However, unlike the Cas9 based system, the Cas3 based system does not only cleave double stranded DNA, but also degrades longer strands of DNA [1].

Generally, CRISPR/Cas-based defence relies on the recognition of known pathogenic DNA sequences (e.g., bacteriophages) via the crRNA (crisprRNA). After the primary invasion of a bacteriophage, parts of the foreign DNA are cleaved [2,3,4,5]. These parts are subsequently integrated as short spacers between repeating palindromic sequences within the CRISPR locus of the bacterial genome, resulting in the formation of CRISPR arrays transcribing for crRNA [2,3,4,5]. Upon reinfection with the same bacteriophage, the transcribed crRNA guides the surveillance complex Cascade (CasABCDE) to its complementary sequences located within the viral DNA [2,3,4,5].

As a consequence, the targeted bacteriophage DNA is unwound, generating the so-called R-loop. The Cas3 helicase-nuclease protein attaches to this R-loop and unidirectionally degrades the invader DNA in a 3′–5′ direction, beginning at the site of a protospacer adjacent motif (PAM) by inducing a sequence of cleavage by a reeling motion [2,3,4,5]. The degradation is facilitated by the large Cas3/Cascade complex, which pulls the substrate DNA towards itself, introducing single strand breaks [2,3,4,5]. The resulting degradation products are approximately the length of a spacer sequence, whereas the cuts are enriched in PAM-like NTT motives at the 3´-end. Therefore, most of the degradation products are suitable for the integration into the CRISPR locus [2,3,4,5].

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Figure 1: General overview over the CRISPR/Cas system. Foreign DNA is taken up and a short spacer sequence cut out and integrated into the CRISPR array in the genome. This array is transcribed together with cas genes. The array is cleaved into several specific gRNAs that form a complex with the cas proteins. These complexes bind to their target sequence and the cas protein cleaves the foreign DNA.

Our Application

Based upon a design kindly provided to us by Dr. Pengfei Xia, we built a chassis that relies on a genetic circuit. The circuit uses the CRISPR/Cas3 complex as a kill-switch by regulating its expression under various environmental conditions. The regulation of our kill-switch is based on three NOT gate modules [14] (see Fig. 2), consisting of doubly negated sets of biosensors chosen due to their ability to recognize the human intestines as a permitted environment. A NOT gate module is a circuit structure, which initiates the death of the bacteria if a certain survival signal is not present.

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Figure 2: Simplified figure of the principle NOT gate modules making up the kill-switch. Modified from Pengfei Xia [14]. The NOT gate modules determine that if a survival signal is not present, the bacteria is killed.

NOT gate module Survival Signals

In our design, common conditions of human intestines were chosen as NOT gate module survival signals (see Fig. 2). Module (1) is determined by a permitted temperature of 37°C, while module (2) requires fatty acid availability in form of Acyl CoA as survival signal. Module (3) is designed to sense N-Acetyl-Glucosamine-6-phosphate (GlcNAc6P) , which is released through the metabolization of mucus by commensal microorganisms [6].

Consequently, if the chassis that carries the kill-switch is in an environment with conditions corresponding to those in the intestine, the Cas3 protein and Cascade cannot be expressed and the CRISPR arrays (crRNA) for the self-targeting of the plasmid and genome are not transcribed (see Fig. 3).

However, if the environmental conditions change, for instance when the bacterium is excreted, the NOT gate modules of the Cas3 system will facilitate the killing of the bacterium. This will in the end result in the degradation of the genomic and plasmid DNA.

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Figure 3: Schematic depiction of the NOT gate modules of the regulator system, which controls the expression of the Cas3 system. (1) The temperature sensing system regulates the expression of cas3 and casABCDE (Cascade). (2) The Acyl-CoA sensing system regulates the expression of the plasmid’s self-targeting array (crRNA). (3) The N-Acetyl-Glucosamine-6 Phosphate sensing system regulates the expression of the genomic crRNA.

(1) Temperature sensing with a permissible temperature of 37°C

constitutively active promoter expresses Clts, a temperature sensitive cI repressor found in the lambda phage. At temperatures significantly below 37°C, Clts is an active repressor of the cI lambda promoter (PClts). This promoter controls the expression of gene encoding for a repressor protein, AraC. Therefore, if the temperature in the bacterium’s environment is below 37°C, Clts is stable and represses the expression of the araC-gene. Therefore, the pBAD promoter, repressed by AraC, is active. As a result, Cas3 and CasABCDE are expressed, allowing for the kill-switch induction (see Fig. 3.1).

Accordingly, if the bacterium is within the body and the temperature is at 37°C, Clts is unstable and cannot repress the expression of the araC-gene. AraC consequently inhibits the pBAD promoter and the CRISPR enzymes for the kill-switch are not available. The Clts and the following FadR sensing system was used by the NTU Taida’s iGEM Project in 2012 [7].

(2) Fatty acid availability inhibiting the plasmid self targeting array

The intake of a fatty meal increases the fatty acid availability within the body, and therefore their metabolite Acyl-CoA’s concentration increases. Long chain Acyl-CoA binds constitutively produced FadR and inhibits its activity [8,9]. FadR represses the promoter PFad, which regulates the expression of the LsrR repressor gene. The LsrR repressor subsequently inhibits the PLsrR promoter [10], regulating the transcription of the plasmid self targeting array.

If the bacterium leaves the body, fatty acids (Acyl-CoA) will not be available. This allows for the activity of FadR, which inhibits PFad. Thus, there will be no LsrR, the self targeting array will be active and used by the Cas3 complex to target the plasmid for degradation (see Fig. 3.2).

(3) N-Acetyl-Glucosamine-6 Phosphate of metabolized mucin inhibits the genomic self-targeting array

Commensal bacteria in the gut metabolize the mucus within the intestines, which increases the level of GlcNAc within the lumen [11]. GlcNAc is taken up by the bacteria through their PTS system and metabolized into GlcNAc-6-P, which binds the repressor protein NagC. When NagC is bound to GlcNAc-6-P, it loses its ability to bind DNA, and therefore its respective regulation activity. In our case, NagC can consequently not serve as a repressor of the nag Operon nagBACDE anymore [12, 13]. Thus, a repressor protein encoded downstream of the promoter, the Lambda phage’s Mnt repressor, can be expressed. Mnt inhibits the genomic self targeting arrays’ transcription by binding the Mnt promoter upstream of the arrays (see Fig. 3.3). The genomic DNA, therefore, cannot be degraded in the presence of GlcNAc-6-P.

Nonetheless, if the bacterium leaves the body and the human microbiome, its GlcNAc-6-P sources will be depleted and the NagC repressor will remain active. As a result, the expression of the MntR is repressed, allowing the transcription of the CRISPR array targeting the genome for degradation.

Advantages of a kill-switch in our project

The incorporation of the Cas3 system into our probiotic bacterium ensures that, once the probiotic bacterium leaves its designated environment, all genetic information is degraded. By using a targeting array for the bacterium’s genome and one for the plasmid containing the gene of interest, the spreading of engineered nucleic acids is prevented, allowing for a safe therapy.

Moreover, this chassis can serve as a foundation for other applications, since it creates a biosafe probiotic bacterium that can be modified by exchanging the gene of interest without losing its unique safety standards.

Lab Work

To begin our work on the CRISPR/Cas3 system we were kindly provided with isolated genomic DNA (gDNA) of E. coli by Dr. Pengfei Xia. We designed PCR-Primers to amplify the cas3 (BBa_K3096001) and Cascade (BBa_K3096007) from the genomic DNA, using Q5 Polymerase. Afterwards, we confirmed the coding sequences via the fragment size using agarose gel electrophoresis (Fig. 3). The amplification of Cascade was not successful, and therefore repeated (Fig.4), which yielded positive results.

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Figure 4: 17/10/19 Gelelectrophoresis of Cas3 and Cascade PCR. Gel loaded (from left to right): Ladder 1kb, Cas3 (72°C), Cas3 (68°C), H2O, Cascade (72°C), Cascade (68°C), H2O. It shows positive PCR of Cas 3 (68°C), expected at ~2.7 kb.
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Figure 5. 07/11/19 Gelelectrophoresis of Cascade PCR. Gel loaded (from left to right): Ladder 1kb, Cascade (68°C), Cascade (64°C). It shows positive PCR of Cascade (68°C), expected at ~4.4 kb.
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Figure 6: 08/15/19 Digestions after Miniprep. Gel loaded (from left to right): Ladder 1kb, psB1C3_K6 1, empty, psB1C3_K6 2, psB1C3_K3 1-2, K10_Cas3 1-2, K10_Cascade 1-2, BBa_R0073(Mnt)_RFP 1-4. K6 expected at ~2 kb and 1.5 kb, K3 expected at ~2 kb and 1 kb, Cas3 expected at ~2.4 kb, ~2 kb and ~0.3 kb, Cascade expected at ~5.1 kb, ~1,2 kb and ~0.3 kb, RFP expected at ~2 kb and ~1 kb.

Next, both cas3 and cascade were successfully ligated with the pBAD promoter of BBa_I13453 to regulate their expression via our temperature sensing system. Identity was confirmed via a digestion (Fig. 4, “K10_Cas3 1-2”, “K10_Cascade 1-2”).

The CRISPR arrays that would target on the one hand the genomic DNA (BBa_K3096014) and on the other hand our GOI-plasmid DNA (BBa_K3096052) had to be newly designed by us and were synthesized together with their respective promoter. The genomic targeting array is controlled by Mnt promoter (BBa_K3096051) and the plasmid targeting array is controlled by the Lsr promoter (BBa_K3096053).

To have a read out for the functionality of our regulator system, we used three reporter plasmids. Two of which were already in the iGEM registry: pLsrR-YFP (BBa_K117008) and pBAD-GFP (BBa_K584000). The last reporter, pMnt-mRFP (BBa_K3096025) was designed and finalized by us (confirmed in Fig. 4 “BBa_R0073(Mnt)_RFP”). Upon cotransformation with the respective regulatory systems, the fluorescence signal would correspond to CRISPR array/Cas3 and Cascade activity.

As far as our laboratory progress is concerned, the CRISPR/Cas3 system parts, as well as their reporter plasmids, were all ready to be tested individually for functionality. However, the regulator system, designed by us using already existing and new biobricks, was not finished yet. Unfortunately, the ligation of at least two parts within each regulatory system was not successful. Consequently, our regulatory plasmids are not functional yet: the constitutive Clts expression with Clts promoter (BBa_K608351) must still be ligated with the araC gene (BBa_K3096002), the constitutive FadR expression and regulated promoter (BBa_K3096005) must be ligated with the lsrR gene (BBa_K091001) and the constitutive NagC expression (BBa_K3096020) must be ligated with the NagC-regulated Mnt repressor gene (BBa_K3096008).

To conclude, while the design for the three final regulator systems, temperature sensitive (BBa_K3096040), Acyl-CoA sensitive (BBa_K3096046) and GlcNAc-6-P sensitive (BBa_K3096048) was completely worked out by us, we did not manage to ligate and create those systems in the laboratory due to several difficulties in restriction digestion and ligation.

Next Steps

As we need more time to generate our regulator systems, we were not able to test and evaluate our CRISPR/Cas3 system parts experimentally. The next upcoming step in the project consequently is the complete cloning of the three regulator systems.

Once this is completed, the functionality of the three systems can be tested by individually cotransforming (different antibiotic resistances) and testing them with the reporter plasmids (fluorescence read-out). If their activity is confirmed, all three regulator systems will be cloned into one plasmid, while the Cas3, Cascade, and CRISPR array sequences will be cloned into a second plasmid.

The system as a whole functioning unit will be cotransformed and proof-of-concept will be approached by analyzing whether the transformed bacteria will kill themselves in prohibitive environments. If the functionality of the kill-switch is confirmed, the next step will be its integration into the genomic DNA of E. coli Nissle 1917, while making sure no antibiotic resistance gene is accidentally integrated, too.

References

  1. “CRISPR-based adaptive immune systems.”, Terns MP,Terns RM, Curr Opin Microbiol 14(3):321–327
  2. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity”, Rodolphe Barrangou Luciano A.Marraffini
  3. “Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3”, Bei Gong, Minsang Shin, Jiali Sun, Che-Hun Jung, Edward L. Bolt, John van der Oost, and Jeong-Sun Kim
  4. Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps", Luuk Loeff, Stan J. J. Brouns, Chirlmin Joo, Molecular Cell
  5. Jackson, Ryan & Lavin, Matthew & Carter, Joshua & Wiedenheft, Blake. (2014). Fitting CRISPR-associated Cas3 into the helicase family tree. Current opinion in structural biology. 24C. 106-114. 10.1016/j.sbi.2014.01.001.
  6. Sicard JF, Le Bihan G, Vogeleer P, Jacques M, Harel J. Interactions of Intestinal Bacteria with Components of the Intestinal Mucus. Front Cell Infect Microbiol. (2017);7:387. Published 2017 Sep 5. doi:10.3389/fcimb.2017.00387
  7. http://2012.igem.org/Team:NTU-Taida/Project/Circuit
  8. https://www.uniprot.org/uniprot/P0A8V6accessed: 19 Jun 2019
  9. Feng Y, Cronan JE. Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PLoS One. (2012);7(9):e46275. doi:10.1371/journal.pone.0046275
  10. Federle MJ. Autoinducer-2-based chemical communication in bacteria: complexities of interspecies signaling. Contrib Microbiol. (2009);16:18–32. doi:10.1159/000219371
  11. Sicard JF, Le Bihan G, Vogeleer P, Jacques M, Harel J. Interactions of Intestinal Bacteria with Components of the Intestinal Mucus. Front Cell Infect Microbiol. (2017);7:387. Published 2017 Sep 5. doi:10.3389/fcimb.2017.00387
  12. Barnhart MM, Lynem J, Chapman MR. GlcNAc-6P levels modulate the expression of Curli fibers by Escherichia coli. J Bacteriol. (2006);188(14):5212–5219. doi:10.1128/JB.00234-06
  13. Konopka JB. N-acetylglucosamine (GlcNAc) functions in cell signaling. Scientifica (Cairo). (2012);2012:489208. doi:10.6064/2012/489208
  14. Peng-Fei Xia, Hua Ling, Jee Loon Foo, Matthew Wook Chang, (2019), Synthetic genetic circuits for programmable biological functionalities