Part:BBa_K3902001

6His-Strep-SUMO-tagged Cas13a with thrombine cleavage site

Cas13a is a DNA-dependent RNAse from the Gram-negative bacterium Leptotrichia buccalis belonging to the class VI CRISPR-Cas systems measuring 1159 amino acids. Its activation by hybridization of the crRNA with which it is associated with its complementary single-stranded RNA allows it to cleave any single-stranded RNA in an aspecific manner, 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 RNA reporter.

This part codes for the Cas13a protein. The Cas13a coding sequence is preceded in 5' by sequences coding for 6 histidine tags allowing the purification of the protein on a nickel affinity column and a Strep-Tag II as well as a sequence coding for a SUMO domain allowing a better solubility of the protein in E.coli. After expression, the 6His-tag and the SUMO domain can be separated from the protein by cleaving the SUMO domain with a SenP2 protein. The 6His-tag can also be separeted alone thanks to a thrombin cleavage site located between the 6His-tag and SUMO loci.

Usage and Biology

Methodology

Production and purification

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

For the production of Cas13a, E.coli Rosetta strain was transformed with plasmids pC0072 LbuCas13a His6-TwinStrep-SUMO-BsaI (Gootenberg 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 SenP2 (Cas13a) or in order to remove the histidine tags and solubilization domains they carried. Cas13a 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 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.

miRNA detection assays

FIGURE 2 : Heparin purification by FPLC results

Cas13a protein coupled with its crRNA had been incubated with bta-miR-7863 or miR-125b miRNAs and a ssRNA reporter. The miRNA hybridized with the crRNA and then activated the Cas13a’RNAse activity allowing it to cleave the reporter which, then, emitted fluorescent signal. Thus, it was possible to follow the detection of the miRNA in a quantitative way by following the fluorescence level.

Results

Production and purification

FIGURE 1 : Coomassie staining (A) and SDS-PAGE gel (B) corresponding to Cas13a and Cas14a1 after digestion with SenP2 (Cas13a) or TEV protease (Cas14a1) and a second purification on nickel column, LA : ladder, NG : Non-digested, FT : Flowthrough, E : Elution (1 or 2), 13 : Cas13a, 14 : Cas14a1

Non-digested Cas13a measuring 155 Kda and digested Cas13a measuring 138 Kda we can conclude that Cas13a has been efficiently digested and its tags and solubilization domains removed.

FIGURE 2 : Heparin purification by FPLC results

Following the purification on heparin column we could observe 1 absorbance peaks at 280 nm at 50 minutes corresponding to Cas13a with tags removed.

miRNA detection assays

FIGURE 3 : Cas13a (cr/miR-125) only activity with different concentrations of crRNAs and times of pre-incubation but constant miRNA concentrations, 10 min of incubation (A), 30 min (B), 60 min (C), 120 min (D)

FIGURE 4 : Initiale velocities corresponding to the results presented on the Figure 5C, Initial velocities (x = a.u./min)

Referring to the results presented in Figures 3 and 4, the optimal conditions for miR-125 detection with Cas13a appear to be a crRNA concentration of 5nM and a 60-minute pre-incubation (Fig 3C and 4). Indeed, although the highest fluorescent signal is observed with a 30-minutes pre-incubation (Fig 3B), the corresponding profile at the other concentrations suggests that this is a result due to measurement or handling error. On the other hand, we can see that a pre-incubation of 10 minutes or 120 minutes seems to result in a low level of fluorescence, even for the most optimal crRNA concentrations (Fig 3A and D).

FIGURE 5 : Cas13a (cr/miR-7863) only activity with different concentrations of crRNAs and times of pre-incubation but constant miRNA concentrations, 10 min of incubation (A), 30 min (B), 60 min (C), 120 min (D)

FIGURE 6 : Initiale velocities corresponding to the results presented on the Figure 7C, Initial velocities (x = a.u./min)

The results presented in Figures 5 and 6 seem to correlate with those presented in Figures 3 and 4. As with miR-125 detection, the conditions for obtaining an optimal signal when detecting miR-7863 by Cas13a appear to be 5nM of crRNA and 60 minutes of pre-incubation (Fig 5C and 6).

FIGURE 7 : Cas13a (cr/miR-125) only reactions with different miRNA concentrations, constant crRNA concentrations (A) and associated initial velocities (B), Initial velocities (x = a.u./min)

The results presented in Figure 7 suggest that the optimal concentration of miRNA for detection of miR-125 by Cas13a is 50 nM. The limit of detection appears to be 20nM.

FIGURE 8: Cas13a (cr/miR-125) only reactions with different bacterial RNA (A) or human RNA (B) concentrations

By observing the results in figure 8 we can conclude that the activation of Cas13a is well specific. Indeed, Cas13 coupled to crRNA-125 does not seem to cleave the fluorophore when incubated with non-specific RNA (bacterial or human) or miR3613.

Conclusions

In conclusion, we succeeded in producing and purifying Cas13a. The tests shows that while the specificity of the Cas13a1 reaction is high as we were able to detect miRNA 125 (Fig 3-5 and 7) and 7863 (Fig 5-6) without detecting non-specific RNAs (Fig 8), the sensibility needs to be improved and incubation times need to be tested over a wider range of durations to further improve the cleaving response. Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1513
Illegal PstI site found at 2368
Illegal PstI site found at 3307
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 64
Illegal PstI site found at 1513
Illegal PstI site found at 2368
Illegal PstI site found at 3307
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 254
Illegal BglII site found at 794
Illegal BglII site found at 1730
Illegal BglII site found at 1940
Illegal BglII site found at 1994
Illegal BglII site found at 2225
Illegal BglII site found at 2498
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1513
Illegal PstI site found at 2368
Illegal PstI site found at 3307
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1513
Illegal PstI site found at 2368
Illegal PstI site found at 3307
Illegal NgoMIV site found at 2446
Illegal NgoMIV site found at 3082
1000
COMPATIBLE WITH RFC[1000]