Sharing has a key role in science. This is why it was important for us to make accessible to other iGEM teams and to the scientific community what we did. Hence, you will find in this page, what we did in this direction.
For the Contribution, we completed the purification protocol and the experimental characterization of BBa_K2306012 and added the data of them to the corresponding BioBricks. All of these may be helpful to other teams. We hope it will make some contribution to the iGEM community.
Contribution to the characterisation of an iGEM part
Part: BBa_K2306012
This cas13a protein is an ortholog of Cas13a from Leptotrichia wadei (LwCas13a), which displays greater RNA-guided RNase activity relative to Leptotrichia shahii Cas13a (LshCas13a) (1). Cas13a is part of the CRISPR (Clustered Regularly interspaced Short Palindromic Repeat) Cas system: Class II, Type VI family of CRISPR-Cas systems. This system is an adaptive bacterial and archaeal defense system protecting the cells from invading phages and other harmful mobile genetic elements. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-nonspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages (2).
Here we report the characterization of a Cas13a enzyme from Leptotrichia wadei. In order to fully characterize the cas13a biobrick, it had to be expressed, purified and tested for its in vitro functionality.
The plasmid pC013 (containing LwCas13a) with a T7 promoter and a N-terminal His-tag were ordered from Addgene in stab culture format transformed into Rosetta (Fig1).
The coding sequence of Cas13a in this biobrick (BBa_K2306012) is not perfectly aligned with the one we purified but this protocol could encompass all cas13 families with an His-tag.
Upstream processing: Expression
Cultivation Procedure
The Cas13a was expressed according to the protein expression protocol on our wiki. To summarize, TB media was inoculated with an overnight culture at OD600 ~0.06 and grown to OD600 ~0.6 in 5L shake flasks at 37 °C with 190 rpm. At this point, the cells were placed on ice for 30 min and induced with 1mM IPTG. The cells were then incubated for 16 hours at 21 °C and 190 rpm, for production of the Cas13a protein. After expression, the cells were harvested using centrifugation at 5000 rpm for 15 minutes and 4°C.
Downstream processing: Purification
Purification Procedure
The processing was adapted from a protocol published by Kellner et al (3): pC013 contains two tags, a SUMO tag and a His tag. We decided to purify the Cas13 only using the Hist tag without cleaving the SUMO tag. And so we developed our own Cas13a protein purification protocol on our wiki. To summarize, the cells were lysed with a high pressure homogenizer in the lysis buffer (20 mM Tris-HCL, 166 mM NaCl, 1 mM DTT, pH 8.0). Once they have been homogenized, the cells are passed through a french press. After clarifying the lysate via centrifugation for 1h at 10 000 rpm at 4°C, the Cas13a protein was purified using an HisTrap HP column followed by size exclusion chromatography.
Results
To check whether we have the protein in the fraction collected from the HisTrap column, we ran an SDS-PAGE gel (Fig2).
As you can see, the fractions are not pure, so we ran a SEC with a membrane of 3 Kda pores: our protein will not be lost because it is bigger than that. We collected the fraction from SEC and ran an SDS-PAGE to view our protein.
The last step was concentrating our protein and achieving a concentration of 2 mg/mL of protein. Then our protein was stored at -80°C
Finally, in vitro functionality was tested with two assays: colorimetric based lateral flow detection assay, and fluorescence-based detection assay to check for proper activity of Cas13a.
In vitro functionality testing: colorimetric based lateral flow detection assay
Assay procedure
The in vitro functionality was tested according to the colorimetric based lateral flow detection assay protocol on our wiki. To summarize, the tube contains the enzyme (Cas13a), the guide RNA (crRNA) and the probes. The probe is a 15 uridine RNA molecule labeled with biotin in 5’ and FAM (6-fluorescein amidite) in 3’. To the reaction tube is added gold nanoparticles coupled with an anti-FITC antibody and reaction buffer. The strip contains two different types of capture molecules: a biotin ligand and an anti-rabbit antibody. Both are fixed on the strip and serve as capture antibodies. The sample is deposited on a sample pad, by capillarity the aqueous solution will migrate. Migrating with the sample the gold nanoparticles are attached to a rabbit anti-FITC antibody which recognizes specifically FITC molecules (Fig4).
Results
We worked on “synthetic sequences” published by Kellner et al. (3). We followed the experimental procedure described in the article. Our goal was to be able to reproduce already published results to set the experimental conditions for the test. A result of such an experiment is shown below (Fig5).
This result approved the experimental conditions as we see a positive read for the synthetic sequence.
In vitro functionality testing: fluorescence-based detection assay
Assay procedure
The in vitro functionality was tested according to the fluorescence-based detection assay protocol on our wiki. To summarize, we designed the cleavage reporter according to Gootemberg et al. in “Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a and Csm6” (4). This reporter is composed of the fluorophore 6-FAM, a poly U sequence and the quencher IABkFQ. We decided to use another quencher called BHQ_1. We ordered this sequence: /56-FAM/rUrU rUrUrU rU/3BHQ_1/ from IDT.
Results
To test the specificity of the Cas13 for detecting genes we designed target sequences where we inserted punctual mutations ranging from 1 to 10 mutations per sequence (Fig6).
Results
We can clearly see that after two mutations we don’t have any detection, so the Cas13 is highly specific (Fig7).
Summary
The Cas13a biobrick was fully characterized, from expression to in vitro functionality testing. The in vitro functionality testing proved that the Cas13a is functional even without cleaving the SUMO tag.
References
1. O. O. Abudayyeh, J. S. Gootenberg, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016). Available from : 10.1126/science.aaf5573
2. Kick, L.M., von Wrisberg, MK., Runtsch, L.S. et al. Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus. Commun Biol 5, 71 (2022). Available from : 10.1101/2021.06.07.447304
3. Kellner, Max J., et al. "SHERLOCK: nucleic acid detection with CRISPR nucleases." Nature protocols 14.10 (2019): 2986-3012 Available from : 10.1038/s41596-019-0210-2
4. Gootenberg JS, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444. Available from : 10.1126/science.aaq0179
Design process of guide RNA and Blast program
In the design page of our project, we completely explain how we designed guide RNA. It is important for us to mention that we did not order them as RNA sequences but that we ordered them as primers. In fact, we proved that it is possible to obtain full guide RNA by PCR. It is something to take into account when we speak about the cost of an experiment. In fact, designing the guide in this way allowed us to have a high amount of guide RNA without spending a lot of money.
Moreover, all the sequences used in our experiments are available on this table. We made the detection of V.aestuarianus and E.Coli accessible and doable by anyone of the future iGEM teams. Finally, to help with the design of the guide RNA, we developed a Blast program to find matches of guide RNA between species. This tool can be useful for the design of guide RNA to detect any microorganism.
Creation of programs for data analysis
Analyzing the data in a proper way is not easy to do especially when we are not specialists of data analysis. This is why we developed several programs to help for the data analysis:
- A script to analyze plate reader data and variants of these scripts according to the different experiments we performed.
- A paper-test quantification algorithm to provide a semi-quantitative readout to paper tests.
Modeling the kinetic of Cas13a reaction
To have a better understanding of the Cas13 kinetic we developed a model which is open source and that can be used by future iGEM teams. The idea is to provide a pipeline code to help with this kind of modeling.
Open source game
To popularize science and synthetic biology to children, we created a board game and decided to make it available everywhere. The game was created in an English version and in a French version. Moreover, in the future, we would like to code this game in HTML to make it available online.
