The end goal of our experimental work was to be able to use the Sherlock technique to detect target genes. For that we devised four main experiments to arrive at our desired outcome:
- Cas13 production and purification
- Guide and target RNA amplification and transcription
- Fluorescence based Shell’Lock
- Paper-based Shell’Lock
Cas13 production and purification
Cas13 production was adapted from a protocol published by kellner et al (1) Cas13, which encompasses four divergent family members (Cas13a–d), is an RNA-guided RNase that produces multiple cleavage sites in single-stranded areas of an RNA target with specific base preferences(1). An essential part of our work was to be able to purify this protein. The plasmid pC013 (containing LwCas13a) were shipped by Addgene in stab culture format (Rosetta).
1.4 L of cultures were grown to an OD600 ~0.6 in TB-media (Cas13a purification protocol) containing 100 µg/mL of ampicillin and induced with 0,5mM IPTG for 16 hours at 21°C. Next, the culture was harvested by spinning down at 5000 rpm for 15 minutes at 4°C, resuspended in a lysis buffer (40ml Tris-HCl (pH 8.0, 1M), 200ml NaCl (5M) and 2ml DTT (1M)), homogenized and sonicated.
The plasmid that we used contained two tags, a SUMO tag, Sumo tag is most frequently used as N-end fusion sequence in yeast to increase the expression and solubility of the desired recombinant protein and an hist tag(2),and his-tag.Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions (3). we decided to purify the cas13 using the hist tag without cleaving the SUMO tag. And so we developed our own protocol for this purification. ( Cas13a Purification protocol) .
We purified this protein first using a histidin trap.
In brief we loaded 10mL of our lysate on the histidin-trap column that was pre-equilibrated with a buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4) We then eluted the protein with a buffer containing Imidazole. We applied several concentrations in a stepwise manner . Our column is equipped with a spectrophotometer to follow UV absorption of eluted proteins. We collected the fractions containing our protein and we ran an SDS-PAGE to check the purity and size of our protein. The next step was to run a dialysis to get rid of the Imidazole. We finally performed a size exclusion chromatography to get rid of remaining contaminants.
We collected the fraction from our 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.
In the end to validate the enzymatic activity of our protein we relied on aSHERLOCK test that we performed with a synthetic RNA target that was used in the paper by Kellner et al (1). We also performed a negative control that does not contain the enzyme. Thus we confirmed that our enzyme is active (Fig.4).
Protocols
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Cas13a Purification Protocol
References
1. Kellner, Max J., et al. "SHERLOCK: nucleic acid detection with CRISPR nucleases." Nature protocols 14.10 (2019): 2986-3012 https://doi.org/10.1038/s41596-019-0210-2
2. https://www.sinobiological.com/resource/protein-review/sumo-tag-purification#:~:text=Sumo%20tag%20is%20most%20frequently,amino%20acid%20sequence%20of%20ubiquitin.
3.https://www.thermofisher.com/fr/fr/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html