Contribution

Project background and overview

We have based our project on the work done by researchers Xu et.al (2021), that created a system called CasMINI from cas12f. They originally designed the casMINI system as a tool to regulate and engineer mammalian genomes. By modifying the protein sequence, Xu et.al (2021) was able to reduce the off-target effects.

We set out to repurpose their casMINI system to function as a diagnostic tool. Specifically, we based our project on a so-called dead CasMINI (dCasMINI). This indicates that the Cas-protein has lost its endonuclease activity to avoid double-stranded breaks in the DNA when it binds. It should also, as indicated by the researchers, be able to bind to its target site more specifically than wild-type cas12f, which we deemed to be a positive trait for the parts used in our project (Xu et.al 2021).

Cas-based systems can be applied to new target sequences of interest. This function is enabled by the single guide RNA (sgRNA) which binds to the nucleic acids of the target with a high degree of specificity and can be easily changed to specify new targets. This means CRISPR-based systems hold potential when applied to diagnostics.

The registered parts are designed to function in a system, where the two dCasMINI complexes composed of 1 of the tagged dCasMINI variants and 1 sgRAN will bind a dsDNA molecule target. See “Parts collection” below for a more detailed description of how the parts are proposed to function as a parts collection.

General

For our diagnostic test, we have utilized the dCasMINI and its re-engineered guide RNA as the detectors. Due to dCasMINI being far smaller in size compared to other available dCas proteins, we chose it as this would help facilitate cloning by not requiring the synthesis of multiple insert fragments. This was based on the experience of one of our supervisors; Athanasios Saragliadis. He made us aware that cloning with multiple gene fragments into a vector simultaniously can have a higher rate of failure than cloning with only plasmid and a single target fragment, and that choosing a Cas-gene capable of being cloned as a single sequence from a single artificially produced DNA sequence (so-called G blocks) would help reduce these problems.

We add this train of thought here, as we believe this could be useful to future iGEM-teams and persons wanting to utilize our engineered protein, or to those wanting to do gene cloning by using artificially synthesized DNA. To facilitate the in vitro use and production of the protein, we codon-optimized it for expression in E. coli. We hope that by contributing this part to the iGEM competition, that this will enable future teams to conduct their own experiments with lower rates of off-site effects.

Xu, X., Chemparathy, A., Zeng, L., Kempton, H. R., Shang, S., Nakamura, M., & Qi, L. S. (2021). Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Molecular Cell, 81(20), 4333-4345.e4. https://doi.org/10.1016/J.MOLCEL.2021.08.008

dCasMINI variant dCas12f

The UiOslo iGEM team did not characterize the pure dCasMINI protein, rather it was used to create two composite parts. These two composite parts His tagged dCasMINI (BBa_K4899006) and Strep tagged dCasMINI (BBa_K4899005) were combined with the corresponding tag. These protein variants may have differ in properties, but their characteristics give an indication about the characteristics of dCasMini itself.

sgRNA

We provide the two sgRNAs used in our test to the iGEM registry the BBa_K4899001 and BBa_K4899002. In both of these are the scaffolds developed by Xu et.al (2021) which can be used by other teams for their own tests. We also contribute the methods we used to develop our version, and how we tried to ensure the chosen anti-repeat region would not interfere with the scaffold of the sgRNA. We hope that other teams will find these methods useful, for an as of the time of writing, poorly characterized and studied guide RNA. Xu, X., Chemparathy, A., Zeng, L., Kempton, H. R., Shang, S., Nakamura, M., & Qi, L. S. (2021). Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Molecular Cell, 81(20), 4333-4345.e4. https://doi.org/10.1016/J.MOLCEL.2021.08.008

Characteristics of dCasMINI – N-terminal His-tag

To the first of our versions of dCasMINI we added a his tag (His-tag Parts Registry ID: BBa_K157011) which was attached to the protein by a linker sequence (Linker Parts Registry ID: BBa_K157013) placed at the N terminal end. We then optimized the expression conditions for the part in a E. coli BL21 DE3 expression system using a pASK-IBA2 vector for which details can be found in the His-tag dCasMINI Part Registry ID: BBa_K4899006, experiments, and expression protocol. We faced problems with the optimization as the dCasMINI was in the insoluble fraction after lysis and centrifugation, we documented the troubleshooting process in our experiments section.

Characteristics of dCasMINI – C-terminal Strep-tag

To the first of our versions of dCasMINI we added a strep tag (Strep-tag Parts Registry ID: BBa_T2012) from the parts registry which was attached to the protein by a linker sequence (Linker Parts Registry ID: BBa_K157013) placed at the C terminal end. We then optimized the expression conditions for the part in a E. coli BL21 DE3 expression system using a pASK-IBA2 vector for which details can be found in the Strep-tag dCasMINI Part Registry ID: BBa_K4899005, experiments, and expression protocol. We faced problems with the optimization as the dCasMINI was in the insoluble fraction after lysis and centrifugation, we documented the troubleshooting process in our experiments section.

We contribute the above parts for applications in various possible diagnostic applications, including the one chosen for our project: endometriosis. The registered parts are designed to function in a complex.

For our project, the proposed use of this parts collection can be summarized as such: “We want to utilize biomarkers in the form of miRNA in the blood of patients with endometriosis to make a lateral flow test for the disease. The miRNA will need to be amplified due to their low concentration in the blood. This amplification process leaves us with dsDNA that our engineered proteins can bind to. These proteins are modified versions of a deadCas system, called dCasMini. It still retains it’s high affinity to nucleic acids, but its endonuclease activity has been lost. The set of two dCasMINI proteins differ in two aspects: they have either an N-terminal his-tag, or C-terminal strep-tag and a chromophore. This enables the proteins to be efficiently purified, bind to our amplified dsDNA product and bind as a complex to a lateral flow test due to their high affinity interaction to our dsDNA via a sgRNA”.