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 (crispr RNA). 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 virus, 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 viral 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].

Common conditions in a healthy human’s intestines include a temperature of 37°C (regulator A), availability of fatty acids in form of Acyl CoA (regulator B) and N-Acetyl-Glucosamin (GlcNAc) (regulator C) (see Figure 1), which is released by the metabolism of mucus by commensal microorganisms [6]. Consequently, if the conditions correspond to those in the intestine, the Cas3 protein and Cascade cannot be expressed and the CRISPR arrays for the self-targeting of the plasmid and genome are not transcribed (see Figure 1 and 2). However, if the environmental conditions change, for instance the bacterium is excreted, the Cas3 system will be activated. Finally, this will lead to the degradation of the foreign plasmid and genomic DNA, killing the bacterium.

The regulation of our kill switch is based on three NOT gate modules, consisting of doubly negated sets of biosensors (see Fig. 2).

A constitutively active promoter expresses Clts, a temperature sensitive cI repressor found in the lambda phage. At temperatures significantly below 37°C (body temperature), Clts is an active repressor of the cI lambda promoter. This promoter controls the transcription of a repressor protein, AraC. Therefore, if the temperature in the bacterium’s environment is below 37°C, AraC is not available and the pBAD promoter, controlled by AraC, is active. Cas3 and CasABCDE are then expressed, allowing for the Kill-Switch induction (see Figure 2.1). Accordingly, if the bacterium is within the body and the temperature is at 37°C, Clts is unstable and cannot repress the expression of AraC. AraC consequently inhibits the pBAD promoter and the enzymes for the self-kill are not available. The bacterium survives. The Clts and following FadR sensing system’s usage was NTU Taida’s iGEM Project in 2012 [7].

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 expressed FadR and hence inhibits its activity [8,9]. FadR represses the promoter pFad, which regulates the expression of the LsrR repressor, which itself represses the the pLsrR promoter [10] regulating the transcription of the plasmid self targeting array. If the bacterium leaves the body, there will be no fatty acids (Acyl-CoA) 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 and degrade it, inducing the degradation of the foreign DNA (see Fig. 2.2).

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, the Lambda phage’s Mnt repressor can be expressed, inhibiting the genomic self targeting array’s transcription which is controlled by the Mnt promoter (see Fig. 2.3). 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, repressing the expression of the MntR, allowing the transcription of the CRISPR array and the Cas3-targeting of the genome, which will kill the bacterium.

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 in order to amplify the Cas3 and Cascade gene from the genomic DNA. We amplified Cas3 (BBa_K3096001) and Cascade (BBa_K3096007) from gDNA of E.Coli MG1655 via PCR (Q5 Polymerase) and checked the fragment size via agarose gel electrophoresis (Fig. 4). The amplification of Cascade was repeated (Fig.5).

Next, both Cas3 and Cascade were ligated into the pBAD promoter of BBa_I13453 so that Cas3 and Cascade could be regulated by our temperature sensing system. 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).

In order to later evaluate whether our regulator system works we also designed three reporter plasmids, two of which were already in the iGEM registry, where each of the three promoters (pBAD, Mnt, Lsr) is upstream of a fluorescent protein. The Lsr (BBa_K117008) and pBAD (BBa_K584000) reporter constructs are already in the iGEM registry, the reporter system for Mnt (RFP expression) was designed by us (BBa_K3096025).

The CRISPR/Cas3 system parts, as well as their reporter plasmids, were all ready to test individually for functionality, however, the regulator system had to be finished first. The above explained regulatory system was fully designed by us with the use of already registered biobricks as well as new parts. Unfortunately, the ligation of the separate parts of our regulator system was not successful (two parts in each system) which meant we were left with non-functional regulator plasmids: constitutive Clt expression with Clt promoter (BBa_K608351), AraC gene (BBa_K3096002), constitutive FadR expression and regulated promoter (BBa_K3096005), LsrR gene (BBa_K091001), constitutive nagC expression (BBa_K3096020), NagC regulated Mnt repressor (BBa_K3096008). 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, however, 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 failed to generate our regulator systems we were not able to test and evaluate our CRISPR/Cas3 system parts experimentally. The next upcoming steps in the project would first be to complete the cloning of the three regulator systems. Once this is completed, the functionality of the three system can be tested by individually testing them with the reporter plasmids (fluorescence read-out) which also confer a different antibiotic resistance. Finally, all three regulator systems will be united on one plasmid as well as the Cas3, Cascade and CRISPR array sequences on a second plasmid. The system as a whole functioning unit will be evaluated by analyzing whether it is able to kill our bacteria and, if successful, it will be integrated into the genomic DNA of E. coli Nissle and evaluated again.