Part:BBa_K3902000

10His-Tagged-Maltose-binding-protein (MBP)-associated Cas14a1 protein with TEV site

Cas12f proteins also called Cas14 are proteins identified mainly in the superphylum of DPANN archaea that can measure between about 400 and 700 amino acids belonging to class V CRISPR-Cas systems. These single-stranded DNA cleaving nucleases are guided by an sgRNA hybridizing to the target DNA. Activation of the nuclease activity of these proteins requires them to dimerize and allows them to cleave single-stranded DNA aspecifically, this phenomenon is called collateral activity. This collateral activity allows the use of this protein for the detection of RNAs using a fluorescent single-stranded DNA reporter.

This part codes for the Cas14a1 protein. The corresponding sequence measuring 1590 bp is preceded in 5' by a sequence coding for 10 histidine tags allowing the purification of the protein on a nickel affinity column as well as a second coding for a Maltose-Binding-Protein (MBP) domain allowing a better solubility of the protein in E.coli.After expression, the 10His-tag and the MBP domain are separated from the protein by a cleavage domain recognized by the TEV protein in order to remove them.

Methodology

Production and purification

FIGURE 1 : Schema representing the purification process of the Cas13a and Cas14a1 proteins

For the production of Cas14a1, E.coli Rosetta strain was transformed with plasmid pLBH531_His10-MBP-Cas14a1 (Harrington et al., 2018). The plasmids, which were ordered from Addgene, carry an ampicillin resistance gene and allow the constitutive production of the transcription inhibitor LacI. Expression of the gene of interest is under the control of the T7 polymerase (already expressed by Rosetta strain) promoter and the lacO operator. Transformed bacteria were grown overnight in LB supplemented with ampicillin (200 mg/mL), chloramphenicol (30 mg/mL) and glucose (0.2%). Cells were diluted in 1 L Terrific Broth (rich medium) supplemented with antibiotics to OD600=0.1 and grown at 37°C until the OD reached 0.5. Then, IPTG (0.5 mM) was added and the culture was shaken at 18°C for 16 h for protein production. The bacteria were harvested by centrifugation at 5000xg for 15 min and resuspended in 40 mL LEW buffer (50mM NaH2PO4 300 mM NaCl pH 8.0) containing Complete protease inhibitor cocktail (Roche). Following lysis by sonication, the proteins carrying His-tags were purified using nickel columns (Protino Ni-IDA 1000, Machery-Nagel) and digested with TEV protease (Cas14a1) in order to remove the histidine tags and solubilization domains they carried. Cas14a1 was then purified by FPLC on heparin column (1 mL, GE Healthcare) using a gradient using solution A (50 mM NaH2PO4 pH 7.5, 100 mM NaCl, 1 mM beta-mercaptoethanol (BME)) and solution B (50 mM NaH2PO4 pH 7.5, 1.5 M NaCl, 10% glycerol, 1 mM BME). Proteins were then diluted 3-fold in 50 mM NaH2PO4 pH 7.5, 20 m M NaCl, 10% glycerol, 1 mM BME. Finally, a Millipore centrifugal device was used to concentrate the protein. Proteins were stored at -20°C after adding glycerol up to 50%. Proteins were analyzed by SDS-PAGE and Coomassie staining. Activity assays

FIGURE 2 : Cas14a-mediated ssDNA activator detection reaction

Cas14a1 protein coupled with its sgRNA had been incubated with a ssDNA activator and a ssDNA reporter. The ssDNA activator hybridized with the sgRNA and then activated the Cas14a1’DNAse activity allowing it to cleave the reporter which, then, emitted fluorescent signal. Thus, it was possible to follow the activation of Cas14a1 in a quantitative way by following the fluorescence level. For the activity assays of the proteins we finally decided to use non-digested Cas14a1.

Results

Production and purification

FIGURE 1 : SDS-PAGE gel corresponding to Cas14a1 after heparin purification, BH : Cas14a1 before heparin purification, LA : ladder, HP : Cas14a1 after heparin purification

The purification of cleaved Cas14a1 was controlled by SDS-Page, unfortunately, the expected sizes before and after digestion are respectively 108 Kda and 65.5 Kda, then, it seems that this purification was not sufficient to purify only the cleaved protein. However, it allowed to remove the fragments corresponding to the tags and solubilization domains.

miRNA detection assays

FIGURE 2 : Cas14a1 (sgRNA and ssDNA activator) only activity with different concentrations of ssDNA activator and times of pre-incubation but constant sgRNA concentrations, 10 min of incubation (A), 30 min (B), 60 min (C), 120 min (D)

Figure 2 seems to show that Cas14a1 exhibits activity even in the absence of the activator. If this activity seems to be increased a bit by its presence, it does not seem to depend on its concentration or on the pre-incubation time.