Parts

• BBa_K1559000 tracr RNA to be used with Cas9
• BBa_K1559001 Cas9 and crRNA array
• BBa_K1559002 rearranged CRISPR/Cas9 system without promoter
• BBa_K1559003 CRISPR/Cas9 system with Anderson high-expression constitutive promoter
• BBa_K1559004 CRISPR/Cas9 system with Anderson medium-expression constitutive promoter
• BBa_K1559005 CRISPR/Cas9 system with Anderson low-expression constitutive promoter
• BBa_K1559006 CRISPR/Cas9 system with pBAD inducible promoter
• BBa_K1559007 CRISPR/Cas9 system with pLac inducible promoter
• BBa_K1559008 CRISPR/Cas9 system with pRha inducible promoter
• BBa_K1559009 Randomized protospacer for CRISPR/Cas9 System
• BBa_K1559010 RFP-Targeting protospacer for CRISPR/Cas9 system
• BBa_K1559011 Low-copy plasmid pTRC99A (trc promoter and lacI removed)

Our assembly strategy involved assembling 4 fragments together (see design page). Over the course of the project, we made these 4 fragments but were unable to assemble them together into one plasmid. We managed to biobrick one of these fragments, the tracrRNA fragment (BBa_K1559000) before the parts submission deadline.

We have also documented and added the sequences of the rest of the parts we designed to the parts registry in the hopes that future iGEM teams may find them useful.

Design

What is CRISPR?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a long name for a characteristic DNA locus that is responsible for the adaptive immunity in most bacteria and in many species of archaea. CRISPRs have been divided into three types (I-III), of which type II is the best understood. A CRISPR type II locus consists of three components: tracrRNA, the Cas genes, and the "array".

Cas gene(s): The core of a type II CRISPR system is Cas9, an endonuclease. Its original purpose in bacteria is to degrade foreign DNA.

Array: By incorporating bits of foreign DNA into this array, the bacteria gains immunity to it in the future. Those bits, which are called spacers, are the instructions that tell Cas9 where to cut. They are complementary to part of the DNA of a previous invader; the exact sequence it matches is called the protospacer. So, the array follows a pattern of spacer - repeat - spacer - repeat, where the repeats are palindromic sequences that aid in identification of the individual spacers. When foreign DNA enters the bacterium, if the bacterium possesses a complementary spacer to it, that spacer is transcribed separately from its surrounding parts as what is called a crRNA.

TracrRNA: Binds to repeats to help with transcription of the array to the currently needed crRNA. crRNA + tracrRNA together is called guideRNA (gRNA) and it is this that directs Cas9 to where it needs to cut.

CRISPR as a plasmid loss genetic safeguard

The CRISPR system is a suitable mechanism for implementing a plasmid loss genetic safeguard because it can cause plasmid loss within the bacteria cell. In particular, the precise targeting of Cas9 to a specific protospacer makes it possible to target the synthetic plasmid only, without causing damage to the cell's own genomic DNA. This is important because if we harmed the host cell in the process of removing the synthetic plasmid, it would be no different from the kill-switch scenario, where there is a fitness cost on cells to have an intact genetic safeguard.

Another incidental benefit of using CRISPR results from the ease with which it can retargetted. By changing the spacer, the inducible CRISPR system we are making for our project can be repurposed by future iGEM teams for applications other than biocontainment.

Existing CRISPR BioBrick

The CRISPR system already exists in the registry as BBa_K1218011, a biobricked version of the pCas9 plasmid described in Jiang et al, 2013. pCas9 was designed to allow expression in E. coli of the type II CRISPR-Cas system originally from Streptococcus pyogenes. It contains in one convenient package the minimum essential components to allow the CRISPR/Cas9 system to work, including the tracrRNA, Cas9 gene, and crRNA array. A useful feature of pCas9 is that the spacer in the CRISPR array has been replaced with a placeholder sequence containing BsaI sites, allowing it to be easily swapped out using the Marrafini lab pCas9 protocol Golden Gate assembly for any desired spacer sequence.

However, pCas9 did not fully meet our requirements because its components are constitutively expressed (see emails from Jiang and Marrafini). In addition, the location of the Cas9 gene in the middle of the sequence makes it difficult to swap out the constitutive promoter for an inducible one.

Re-engineering pCas9

In order to facilitate the inducible expression of Cas9 while retaining benefits of having the essential components of the CRISPR/Cas9 system in one biobrick, we decided to re-engineer pCas9 by rearranging the order of its components. Our new design (BBa_K1559002) puts the Cas9 right at the beginning (upstream end) of the biobrick, so that it is removed from the control of the existing constitutive promoters originally from Streptococcus pyogenes, and is instead exposed to transcriptional control from promoters added upstream of the biobrick.

Figure 2: A schematic of the rearrangement of pCas9 to expose Cas9 directly to the control of an upstream promoter.

Conceptually, this new design could be constructed by dividing the existing pCas9 part BBa_K1218011 in two sections, one containing the tracrRNA and bidirectional constitutive promoter region (BBa_K1559000) and another containing the Cas9 coding sequence and CRISPR array (BBa_K1559001). By joining the Cas9-array section to the upstream end of the tracrRNA section, we would have effectively moved Cas9 to the front of the part (see BBa_K1559002). The expression of the tracrRNA and crRNA array are unaffected because they have not been moved relative to their original promoters.

Testing Cas9 expression with various promoters

In order to test the effect of various levels of Cas9 expression, we planned on putting our reengineered CRISPR/Cas9 system under the control of Anderson constitutive promoters of various strengths (high, medium, low).

We also planned on testing various inducible promoters (pBad, pLac, pRha) with our reengineered CRISPR/Cas9 system.

One problem encountered by previous iGEM teams (eg: Paris Bettencourt 2012) was leaky expression. We will be expressed our CRISPR/Cas9 system in a low-copy plasmid (modified pTRC99A) to reduce the effect of leaky expression. As an extension to our project, we also planned on testing tet-off (BBa_K1061012, BBa_K1061013) as a low leaky expression alternative for the inducible control of the CRISPR/Cas9 system.

Assembly Strategy

We planned on using Gibson assembly to assemble our complete plasmids, including the rearrangement of pCas9 described above, all in one reaction. The four fragments of our assembly are as follows:

1. biobrick prefix, promoter, and rbs (synthesized as gblock, overlaps with C and B)
2. Cas9 and crRNA array (PCR from BBa_K1218011)
3. tracrRNA and biobrick suffix (synthesized as gblock, overlaps with B and D)
4. the low-copy origin of replication and ampicillin resistance marker from pTRC99A (PCR from pTRC99A)

CRISPR activity assay

We designed a CRISPR activity assay order to measure the activity of our CRISPR/Cas9 system under the control of various constitutive and inducible promoters.

This assay involves two plasmids, a "target" plasmid, containing a protospacer and PAM, as well as the CRISPR plasmid itself (promoter, cas9, crRNA array, tracrRNA), with the corresponding spacer sequence inserted into the spacer insertion site of the crRNA array. The two plasmids must have different antibiotic selectable markers.

In our case, we chose to use RFP in pSB1C3 as our target plasmid because it has an easily recognizable phenotype (red colonies), as well as a different antibiotic resistance marker (Chloramphenicol resistance) than our CRISPR plasmids (which are Ampicillin resistant). To make our CRISPR plasmids target the RFP plasmid, we found a suitable protospacer within the sequence of RFP to insert into the spacer insertion site of our CRISPR plasmids.

Summary of the two plasmids:

• Target plasmid: constitutively active RFP (red colonies), Chloramphenicol resistance
• CRISPR plasmid: Cas9 (controlled by various promoters), crRNA array containing spacer targeting a sequence in RFP, tracrRNA, Ampicillin resistance.

Before starting our assay, it is necessary to transform the target plasmid (RFP) into the wildtype (MG1655) strain of E. coli. This allows us to obtain a strain of cells known to carry the target plasmid.

For target plasmids that do not exhibit a visible phenotype like red fluorescence, the CFU of cells that retained the target plasmid even after successful transformation of the CRISPR plasmid can be determined by plating on both antibiotics (eg Choramphenicol and Ampicillin). This value can then be divided by the CFU of a plate with only the CRISPR plasmid antibioc resistance (eg Ampicillin only) and subtracted from one to determine the rate of CRISPR activity.

In our assay, these target-carrying cells are transformed with our CRISPR plasmid and plated on agar plates containing Ampicillin (the antibiotic marker for the CRISPR plasmid). Colonies growing on the Ampicillin plates result from cells that were successfully transformed with our CRISPR plasmid. Since all of these cells were known to carry the target RFP plasmid at the start of the assay, we can reasonably conclude that any colonies that are no longer red after being successfully transformed with the CRISPR plasmid had lost the target RFP plasmid due to cleavage by the CRISPR/Cas9 system. The percentage efficiency of the CRISPR/Cas9 system can be determined by dividing the CFU of non-red colonies by the total CFU on the Ampicillin plate.

In the case of inducible promoters, the assay can be repeated with and without the inducer present in the medium, to determine the "on" and "off" rates of CRISPR activity.

Note: Our CRISPR activity assay requires the CRISPR/Cas9 system and the protospacer it targets to be on two different plasmids. However this is not intended to be the actual configuration of our plasmid-loss system. We envision our final product to be a plasmid on which the CRISPR/Cas9 system, controlled by an inducible promoter, and the protospacer targeted by the CRISPR are both present on the same plasmid.

References

1. Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233 - 239. doi:10.1038/nbt.2508