Part:BBa_K4611010

LwaCas13a

Cas13 orthologs, the single effectors of the Class 2 type VI clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) systems, are RNA-guided ribonucleases1. The CRISPR RNAs (crRNAs) of this family contain a direct repeat stem loop that interacts with the Cas13 protein to form an RNase-inactive binary complex and a spacer sequence that base pairs with the target RNA2. The resulting Cas13–crRNA–target ternary complex undergoes a large-scale conformational change in which two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains move toward each other to form a single catalytic pocket to cleave the target RNA. Intriguingly, this catalytic pocket localized on the outer surface of the target-activated Cas13 complex can non-specifically cleave any surrounding RNA molecules in a characteristic ‘collateral effect’2. This target-triggered collateral activity, originally an immune defense mechanism intended to induce host dormancy and prevent the propagation of invading phages, has been rapidly developed for in vitro detection of nucleic acids3.(Yang J...2023) LwaCas13a is a kind of Cas13 protein from Leptotrichia wadei.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2830
  Illegal PstI site found at 2851
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2830
  Illegal PstI site found at 2851
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2830
  Illegal BglII site found at 1286
  Illegal BglII site found at 1496
  Illegal BglII site found at 1763
  Illegal BglII site found at 2528
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2830
  Illegal PstI site found at 2851
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2830
  Illegal PstI site found at 2851
  
• 1000
  COMPATIBLE WITH RFC[1000]

JU-Krakow 2024 Contribution

Introduction

This DNA template contains the coding sequence (CDS) of the LwaCas13a protein, which can be utilized within the SHERLOCK system for the specific detection of RNA (or DNA with an additional in vitro transcription step). [1] 

Most often not only CDS but also additional tags are appended to the protein’s sequence and expressed. The tags facilitate protein’s purification process and/or solubility. By providing specific cleavage sites the tags may be cut off to obtain native protein conformation. 

Sequence source

In case of our experiments the pC013 [2] plasmid was used (it is often used in other LwaCas13a purification protocols too) – it contains the CDS of the LwaCas13a protein with appended 6xHis-TwinStrep-SUMO tag at the N-terminus of the protein. The tag may be cleaved off by utilizing the SUMO protease. 

Our contribution to the BBa_K4611010 part 

• Detailed LwaCas13a purification protocol

• Comparison of the tagged and untagged LwaCas13a in terms of thermal stability (NanoDSF), structure (Circular Dichroism) and activity (in SHERLOCK reaction) to assess the need for tag removal

Biology&Usage [1,2,3]

The LwaCas13a protein is a member of the Cas protein family. In addition to the cis-cleavage activity that is characteristic of most Cas proteins, such as Cas9, which cleaves target nucleic acids, Cas13 proteins exhibit a non-specific collateral activity that indiscriminately cleaves any single-stranded RNA in proximity to the protein. To activate this collateral activity, the CRISPR RNA (crRNA) must bind to the target RNA, which subsequently triggers the protein's nucleolytic domain. This mechanism is harnessed in the SHERLOCK method, where the cleavage of a reporter generates a signal—either fluorescence or a specific binding on a lateral flow assay (LFA) strip—indicating the presence or absence of the target nucleic acid.

Figure 1. The SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) Method Mechanism

Protein purification protocol optimization

The 6xHis-TwinStep-SUMO tag that is bound to the protein (when pC013 plasmid is used) facilitates its purification. In scientific literature, numerous procedures for purifying the Cas13 proteins (most often LwaCas13a) have been described. Often, those involve a single-step purification using IMAC (Immobilized Metal Affinity Chromatography) [4], [5], [6], sometimes with an additional SEC (Size Exclusion Chromatography) step [7]. Another approach involves a three-step purification process – affinity chromatography using a streptavidin [1] or histidine [8] tag (IMAC) followed by IEC (Ion Exchange Chromatography), finishing with SEC (Size Exclusion Chromatography).

In most protocols, the 6xHis-TwinStrep-SUMO tag located at the N-terminus of the protein is cleaved off [1], [5], [6]. No data justifying the removal of the tag, which requires the use of the relatively expensive SUMO protease, was found in the literature.

Our study focuses on optimizing the purification protocol to obtain the purest, active LwaCas13a protein with minimal labor and cost.

Based on literature research (mostly on Kellner’s et. al. publication [1]) and our instructors’ experience, we created our own protocol for LwaCas13a purification, which can be accessed here..

Below, we have outlined the most important steps of the protein production and purification process, along with comments about their purpose for the experiments.

1. Protein Expression and Genetic context:

• System: Proteins were expressed in E. coli strain Rosetta 2(DE3)pLysS using the pET system.

• Significance: This setup supports high-level expression of recombinant proteins, essential for obtaining sufficient quantities for purification.

2. Bacterial Cell Disruption:

• Method: Sonication was used to lyse the bacterial cells.

• Significance: This method effectively disrupts the cells, releasing the expressed proteins into the supernatant for further purification.

3. Three-Step Purification Process

• IMAC (Immobilized Metal Affinity Chromatography):

  • Objective: To evaluate whether the HisTag can be as effective as the StrepTag in preserving protein purification efficiency.

  • Results: IMAC was successful in reducing contaminants and concentrating Cas proteins in collected fractions, as demonstrated by SDS-PAGE (Figure 3).

  • Elution Optimization: Testing of 150 mM and 300 mM imidazole concentrations revealed that 300 mM is more effective in eluting all bound protein (Figure 4)

  • Tag Removal and Analysis: Half of the fractions were treated with SUMO protease to remove the 6xHis-TwinStrep-SUMO tag. The goal was to compare the activity, structure, and stability of the tagged versus untagged proteins (after all purification steps).

    

    Figure 2. Schematic depiction of the LwaCas13a protein with and without the appended tag.

• IEC (Ion Exchange Chromatography):

  • Objective: To remove remaining protein contaminants and concentrate the sample before final purification.

  • Confirmation: Successful removal of contaminants was confirmed by SDS-PAGE (Figure 5).

• SEC (Size Exclusion Chromatography):

  • Objective: To achieve highly pure Cas13a protein free of contaminants.

  • Confirmation: Purity was validated by SDS-PAGE (Figure 8).

4. Comparative Analysis of Tagged and Untagged Protein

• Circular Dichroism (CD):

  • Purpose: To compare the secondary structure of the proteins with and without the tag (Figure 11).

• Nano Differential Scanning Fluorimetry (NanoDSF):

  • Purpose: To assess thermal stability (Figure 12).

• Activity Tests in SHERLOCK:

  • Purpose: To compare activity and check for non-specific interactions or constitutional collateral activity using synthetic DNA from Kellner et al. [1] (Figure 10).

Conclusion:
The optimized protocol, without the tag cleavage step, enabled the production of highly pure and active Cas13a, suitable for SHERLOCK detection assays. The tag cleavage step turned out to be unnecessary as no differences in terms of protein activity, stability and structure were observed.

Experiments & Results

IMAC

Goals

Contamination reduction and Cas concentration in protein samples.

Methods

The first stage of protein purification was carried out due to the presence of an N-terminal histidine tag, which binds to the nickel resin column bedding. The process was conducted at 4°C. 

The column with packed resin was washed with the NI buffer (flow rate 3 ml/min, 5 column volumes (cv)), the sample (supernatant after sonication and centrifugation) was loaded (flow rate 1.5 ml/min), and the column was washed with 5 cv of NI buffer (flow rate 3 ml/min) to remove unbound residues. 

A two-step elution was performed: a buffer prepared by mixing NI and I buffers in a 1:1 ratio with a final imidazole concentration of 150 mM, followed by buffer I containing 300 mM imidazole. 15 fractions of 2 ml in each elution step were collected.

Notes:

• Buffer without imidazole (NI): 0.5 M NaCl; 20 mM Na₃PO₄; 5% glycerol, 1 mM DTT, protease inhibitors. pH=8.0 

• Buffer with 300 mM imidazole (I): 0.5 M NaCl; 20 mM Na₃PO₄; 5% glycerol, 1 mM DTT, protease inhibitors, 300 mM imidazole;. pH=8.0 

• (Before usage of NI and I buffers add 1 cOmplete Ultra per 30 ml of buffer. It has the protease inhibitors listed in the buffer composition.)

Results

Figure 3. SDS-Page gel for LwaCas13a - samples from expression and IMAC purification. Height on which Cas13a should be observed is indicated by purple arrows.

Wells key: unind. – uninduced bacterial culture, ind. – culture after IPTG induction, sonic. – supernatant after sonication and centrifugation, prec. – sediment after sonication and centrifuging, FT – flow-through from sample application on His-resin column, wash – flow-through from column washing before elution; E1, E2, E3… - fractions eluted with 150 mM imidazole buffer, *E1, *E2, *E3 - fractions eluted with 300 mM imidazole buffer

Figure 4. Total eluted protein concentrations in fractions after IMAC (Cas13a).

Conclusions:

1. IMAC was effective in partially purifying the Cas protein - percentage content of contaminants in the solution decreased. Although they are still present in significant amounts.

2. Fractions were enriched with the produced protein (thicker bands corresponding to the produced protein – marked by arrows on Figure 3)

150 mM imidazole elution is efficient, however small portion of protein is eluted by 300 mM – in the next purification it would be advised to elute protein using 300 mM imidazole (Figure 3, 4)

SUMO protease digestion

Goals

Divide Cas13a into two pools – SUMO protease digested and undigested (= without and with 6xHis-TwinStrep-SUMO tag) to compare their activity, structure and stability (after final purification).

Methods

10 ml of the selected samples for Cas13a (samples 1,3,5,7,9) were diluted to 30 ml with buffer A, 10 μl of DTT (1 M), 60 μl of NP-40 detergent and 50 μl of SUMO protease (1 U/μl) were added. The mixture was digested overnight on a rocker at 4°C.

• Buffer A: 20 mM Na₃PO₄; 5% glycerol, 1 mM DTT. pH=7.5

IEC

Goals

Further removal of protein contaminants and concentrating the Cas protein sample before final purification using SEC

Methods

This purification stage was conducted separately for two samples: the digested protein (with a theoretical pI of 9.72) and the undigested protein (pI 9.62).

The separation was performed using an ӒKTA Pure FPLC (Fast Protein Liquid Chromatography) system and a cation exchange column Mono S 4.6/100PE (Cytiva) with a volume of 1.7 ml.

The device was programmed based on the mentioned Kellner’s protocol [1], starting with preequilibrating the column with 12.5% buffer B (5 cv). The sample of the undigested protein was diluted with buffer A (pH 7.5) to 30 ml. Sample was loaded, followed by washing the column with 12.5% buffer B (5 cv), eluting the bound protein with a 12.5%-100% buffer B - 10 cv, collecting fractions of 2 ml each. Followed by washing the column with 100% buffer B (10 cv), and equilibrating with 12.5% buffer B (5 cv). Flow-rate on all steps: 1 ml/min

The procedure was repeated for the digested protein sample with following changes for elution: 12.5-80% buffer B, volume of collected fractions = 0.5 ml.

• Buffer A: 20 mM Na₃PO₄; 5% glycerol, 1 mM DTT. pH=7.5

• Buffer B: 2 M NaCl; 20 mM Na₃PO₄; 5% glycerol, 1 mM DTT. pH=7.5

Results

Figure 5. SDS-Page gel for LwaCas13a - samples after IEC. Cas13a with the tag is pointed to by a purple arrow, without - by a blue arrow. The purple rectangle marks two bands that correspond to the cleaved 6xHis-TwinStrep-SUMO tag.

Wells key: entry – pooled fractions after IMAC, sample appl. – flow-through from sample application on the column, wash – flow-through from column washing; E1, E2, E3… - eluted fractions, numbers correspond to the numbers of fractions on chromatograms (Figure 6, Figure 7). Samples with * correspond to samples after SUMO protease digestion.

Figure 6. Chromatogram. Cas13a IEC for SUMO protease undigested sample.

Figure 7. Chromatogram. Cas13a IEC for SUMO protease digested sample.

For both SUMO protease digested and undigested samples reduction in protein contaminants, approximately 2-fold, can be observed, although they still remain in significant amounts. Concentration of Cas13a in the collected fractions is visible (Figure 5). The elution maximum occurs at approximately 35% buffer B in A, which corresponds to 0.7 M NaCl. 

Using a cation-exchanger was a good decision to separate the cleaved tag (marked with purple rectangle on Figure 5).The pI of 6xHis-TwinStrep-SUMO tag is 6.57 which results in no binding of the tag to the column bedding.

Protein samples concentration

Goals

Reducing the volume of the sample to inject it into the SEC column.

Methods

Samples 4-6 (undigested) and 9-20 (digested) were concentrated in a centrifugal concentrator to a volume of approximately 0.5 ml (centrifugation parameters: 15 min, 4 000 g, 4°C).

Results

The sample volume for both proteins was reduced to approximately 0.5 ml.

SEC

Goals

Obtaining pure LwaCas13a.

Methods

SEC was performed on the same device as IEC (flow rate 0.5 ml/min). The Superdex 200 10/300 GL (Merck) column was equilibrated with buffer S (1 cv), the concentrated sample of undigested protein was loaded and eluted, collecting 0.5 ml fractions (1 cv), then the column was washed and re-equilibrated (1 cv). SUMO digested sample was loaded.

• Buffer S: 50 mM Tris-HCl (pH 7.5); 0.6 M NaCl; 5% glycerol. pH=7.5

Results

Figure 8.SDS-Page gel for LwaCas13a - samples after SEC. Cas13a with tag is pointed to by a purple arrow, without - by a blue arrow.

Wells key: E1, E2, E3… - eluted fractions, numbers correspond to the numbers of fractions on chromatogram (Figure 9). Samples with * correspond to samples after SUMO protease digestion

Figure 9. Chromatogram Cas13a. SEC. First sample: SUMO protease undigested, second: digested.

Conclusions

We successfully conducted a whole protein production and purification process for the LwaCas13a protein. However, 2 bands for SUMO protease digested samples are present (Figure 8) due to the protein not being fully digested. The higher band (purple arrow) corresponds to the protein with the 6xHis-TwinStrep-SUMO tag, and the lower band (blue arrow) to the protein without the tag.

Preparation for protein tests

Following fractions were pooled:

1. Undigested: 17-20 (0.5 ml each; together 2 ml), protein concentration measured using the Bradford method: 0.73 mg/ml. Total LwaCas13a mass: 1.46 mg

2. Digested: 40-44 (0.5 ml each; together 2.5 ml), protein concentration measured using the Bradford method: 0.87 mg/ml. Total LwaCas13a mass: 2.175 mg

SHERLOCK activity test

Goal

Comparing activity of LwaCas13a with and without the 6xHis-TwinStrep-SUMO tag.

Methods

SHERLOCK was conducted as described in Kellner’s protocol [1].

Results

Figure 10. Activity comparison of LwaCas13a with and without the 6xHis-TwinStrep-SUMO tag.

Figure key: 

• 1 – SUMO protease digested LwaCas13a added to SHERLOCK mix

• 2 – SUMO protease undigested LwaCas13a added to SHERLOCK mix

• 3 – water added instead of Cas13a (negative control)

• 4 – RNase A added instead of Cas13a (positive control)

• 5 –target RNA and crRNA replaced with water in SHERLOCK mix, for SUMO digested LwaCas13a

• 6 – target RNA and crRNA replaced with water in SHERLOCK mix, for SUMO undigested LwaCas13a

Conclusions

Produced LwaCas13a is active. No statistically significant differences in activity are observed when comparing SUMO protease digested (1) and undigested (2) samples. The protein does not present constitutive collateral activity, as significantly lower signals are observed for probes without target and crRNA (5-6). Produced LwaCas13a can be used in further SHERLOCK tests.

Circular Dichroism (CD)

Goal

Comparing structure of both proteins with and without the 6xHis-TwinStrep-SUMO tag.

Methods

Measurements were conducted using a Jasco J-710 spectropolarimeter.

Measurement parameters: wavelength range: 190-250 nm, resolution: 1 nm, scanning mode: continuous, scanning speed: 20 nm/min, response time: 4 s, bandwidth: 2.0 nm. Five data series were collected. Measurements were performed in a quartz cuvette with an optical path length of 0.1 mm.

Results

Figure 11. Dependence of mean residue molar ellipticity for LwaCas13a with and without tag in the far-UV range.

Table 1. Proportions of various secondary structure types determined from Circular Dichroism spectra for LwaCas13a.

Conslusions: Mean residue ellipticity spectra (Figure 11) overlap and the proportions of the individual secondary structures in the protein versions (Table 1) do not differ significantly. This points out that the tag’s presence does not have significant impact on proteins’ structure, therefore should not affect proteins function what has been earlier confirmed in the activity tests.

NanoDSF

Goal

Comparing thermal stability of both proteins with and without the 6xHis-TwinStrep-SUMO tag.

Methods

Measurements were conducted using the Prometheus NT.48 device (NanoTemper Technologies).

Parameters: heating range from 20-95°C at a rate of 1°C/min. Measurements were performed in triplicates.

Results

Figure 12. Dependence of first derivative of the fluorescence intensity change from temperature for LwaCas13a. D – sample digested using SUMO protease, UD – sample undigested

Conclusions

Curves corresponding to the digested (D) and undigested (UD) proteins overlap. This proves that the tag’s presence does not have significant impact on protein thermal stability, which additionally justifies keeping the 6xHis-TwinStrep-SUMO tag attached.

FINAL CONCLUSIONS

1. We recommend purifying LwaCas13a according to our protocol, which can be accessed here.

2. From sediments that we used for protein purification we achieved:

   • 1.46 mg tagged LwaCas13a – enough for 11,500 reactions according to Kellner’s protocol [1]

   • 2.175 mg untagged LwaCas13a – enough for 17,000 reactions

Therefore, it is justified to conduct a longer and more costly protocol (than 1-step purification protocols suggested in some publications [4], [5], [6]) to obtain the purest possible Cas protein. This should contribute to greater reliability of the results of SHERLOCK tests determining the presence of nucleic acids in the analyzed samples. A lower actual amount of protein than assumed (because of other contaminating proteins present in the preparation) would lead to the cleavage of fewer reporters than at the correct protein concentration, resulting in an underestimation of the detected nucleic acid concentration. This could even contribute to false-negative results if the detected genetic material is present in low amounts in the sample and the fluorescent signal does not exceed a certain threshold value.

3. IMAC can effectively replace StrepTag affinity chromatography suggested by Kellner [1]

Comparing the amount of LwaCas13a that we purified, we can even state that IMAC is more effective than (or at least equally effective as) StrepTag chromatography. We obtained 3.635 mg of pure Cas13a from 1.5 L of large-scale culture, whereas Kellner [1] reports 0.5-1 mg from 4 L culture.

4. Not cleaving off the 6xHis-TwinStrep-SUMO tag is justified by activity tests (Figure 10), CD (Figure 11, Table 1) and NanoDSF (Figure 12).

   Resources

   [1] M. J. Kellner, J. G. Koob, J. S. Gootenberg, O. O. Abudayyeh, and F. Zhang, “SHERLOCK: nucleic acid detection with CRISPR nucleases.,” Nat Protoc, vol. 14, no. 10, pp. 2986–3012, Oct. 2019, doi: 10.1038/s41596-019-0210-2.

   [2] J. S. Gootenberg et al., “Nucleic acid detection with CRISPR-Cas13a/C2c2,” Science (1979), vol. 356, no. 6336, pp. 438–442, Apr. 2017, doi: 10.1126/science.aam9321.

   [3] O. O. Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.,” Science, vol. 353, no. 6299, p. aaf5573, Aug. 2016, doi: 10.1126/science.aaf5573.

   [4] A. S. Savinova, E. Yu. Koptev, E. V. Usachev, A. P. Tkachuk, and V. A. Guschin, “Cas13a: purification and use for detection of viral RNA,” Bulletin of Russian State Medical University, no. 2, pp. 21–25, 2018, doi: 10.24075/brsmu.2018.021.

   [5] B. An et al., “Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification,” Front Microbiol, vol. 12, Oct. 2021, doi: 10.3389/fmicb.2021.732426.

   [6] H. Khan et al., “CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2,” Chinese Chemical Letters, vol. 30, no. 12, pp. 2201–2204, Dec. 2019, doi: 10.1016/j.cclet.2019.10.032.

   [7] Montpellier 2022 iGEM Team, “Cas13a Purification Protocol.” Accessed: Jun. 05, 2024. [Online]. Available: https://2022.igem.wiki/montpellier/results

   [8] A. East-Seletsky et al., “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection,” Nature, vol. 538, no. 7624, pp. 270–273, Oct. 2016, doi: 10.1038/nature19802.

   

   End of JU-Krakow 2024 Contribution