Part:BBa_K4170014

SUMO-LbuCas13a with 6XHis purification tag

This plasmid contains the coding sequence (CDS) of codon-optimized SUMO-LbuCas13a protein for recombinant LbuCas13a protein expression in E.coli in fusion with the 6xHis affinity tag to facilitate the efficient purification of the protein utilizing a Ni-NTA affinity purification methodology. In addition, the codon-optimized LbuCas13a protein is expressed in fusion with small ubiquitin-like modifier (SUMO) protein which favors the increased expression and purificitaion of the recombinant LbuCas13a protein from the soluble cytoplasmic function. This part is an "improved" version of the Cas13a Lbu part (BBa_K2926001) deposited from iGEM19_Bielefeld-CeBiTec team in the registry. Specifically the coding sequence of the SUMO-LbuCas13a is codon optimized for efficient recombinant protein expression in E.coli and an addition SUMO solubility tag has been inserted at the N-terminus of the LbuCas13a to enhance the protein recovery from the soluble cytoplasmic bacterial fraction maintaining its proper folding.

CRISPR/Cas13a systems as RNA sensors

CRISPR-Cas systems are RNA-guided adaptive immune systems that protect prokaryotes from foreign genetic elements derived from evading viruses and phages (Shan et al., 2019). The Cas13a is ribonuclease with a double biological functionality: catalyzes the crRNA maturation and degrades the RNA-guided ssRNA (single-stranded RNA) interdependently using two separated catalytic sites (Rath et al., 2015). Generally, CRISPR/Cas system can be divided into two main classes, class I and II, according to the system comprising a single or multiple effectors (Liu et al., 2017). Among them, class II (e.g., Cas9, Cas12, and Cas13) possesses more widespread application, due to its simple components (a single effector protein and a programmable guide RNA, Wang et al., 2021). Cas13 can be further divided into four subtypes, Cas13a-d, exhibiting diverse primary sequences except the two highly conserved HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains, which are responsible for both cis- and trans-RNase activities (Florczuk et al., 2017). Structural studies revealed that Cas13a adopts a bilobed architecture including recognition (REC) and nuclease (NUC) lobes (Wang et al., 2021, Zhou et al., 2020). CRISPR-Cas12,13,14 exhibits nonspecific degradation of non target (trans cleavage) after specific recognition of nucleic acids, thus CRISPR/Cas biology promises rapid, accurate, and portable diagnostic tools, the next-generation diagnostics. Cas13a due to its propensity to cleave RNAs after binding a user-defined RNA target sequence, is used to detect single molecules of RNA species with high specificity (Liu et al., 2017).

Similarly to Cas9, Cas13a recognizes the short hairpin of the crRNA and made a complex. The target specificity is encoded by the 28-30-nt crRNA spacer sequence which is complementary to the target region. Cas13s exhibit collateral activity and after recognition and cleavage of the target transcript, degrades non-specifically any nearby RNAs regardless of complementarity to the spacer.

Rare codon analysis for expression in E.coli

Utilizing the Rare Codon Analysis tool from Genscript we evaluated the codon usage frequency of the two coding sequences as described in detail below.

Codon optimized SUMO-LbuCas13a (BBa K4170014) for E.coli expression

According to the Rare Codon analysis tool, the codon optimized SUMO-LbuCas13a protein displayes a high CAI value (approximately 0.8). The CAI value is a codon adaptation index and the higher the CAI value the higher the change that the gene will expressed efficiently. The CAI value equal to 0.8 is acceptable ensuring a high change of efficient protein expression. In addition, the percentage of low frequency codons (CFD) based on the E.coli host organism is about 3%. This minimum percentage ensures that the percentage of rare codons in our sequence is extremely low and cannot affect the translational efficiency.

Cas13a Lbu part (BBa_K2926001) deposited from iGEM19_Bielefeld-CeBiTec team

On the other hand, the Cas13a Lbu part (BBa_K2926001) deposited from iGEM19_Bielefeld-CeBiTec team display a relatively low CAI value (approximately 0.65) which deviates from the acceptable limits for efficient protein expression in E.coli. In additon, the percentage of low frequency codons (CFD) is 7%, higher that the codon optimized SUMO-LbuCas13a sequence deposited by : iGEM22_Thessaloniki_Meta. These relativelly low values could reveal the reduced protein expression efficiency in E.coli. However, these values (CAI & CFD) are clearly improved when the S. cerevisiae Yeast Strain is selected as a host organism.

Basic parts of the device

The part sample which is flanked at the beginning and the end with prefix and suffix respectively, is composed of the following basic parts assembled together in series and downstream of the prefix:

6XHis tags: 6xHis affinity tag
Thrombin site: thrombin recognition and cleavage site
bdSUMO CDS: small ubiquitin-like modifier (SUMO) protein
LbuCas13a CDS: Cas13a protein derived from Leptotrichia Buccalis

Figure 1.The SUMO-LbuCas13a sequence in pSB1C3 plasmid backbone. The primers that used for the mutaton of illegal sites by mutagenesis PCR are also depicted

Cloning strategy for SUMO-LbuCas13a assembly with the pSB1C3 plasmid

For the final assembly of the PCR amplified genetic elements into pSB1C3 plasmid we followed the Golden Gate-based ‘SevaBrick Assembly’ method and the SEVA 3.1 platform introduced by Stamatios G. Damalas and colleagues (Damalas et al., 2020).

'SevaBrick assembly' method

The Golden Gate-based ‘SevaBrick Assembly’ method was introduced by Stamatios G. Damalas and colleagues (Damalas et al., 2020). This method consists of standardized primers and protocols and facilitates the straightforward one-step assembly of multiple genetic elements into the SEVA 3.1 or the pSB#X# (# is determined by the identity of the replication origin and the letter X is determined by the antibiotic resistance marker) backbones in a fast and reliable process. The SevaBrick Assembly is a method where all the parts and backbones to be assembled are PCR amplified from any SEVA 3.1 or BioBrick vector, using a core set of standard long primers. All SevaBrick primers anneal on standard sequences of the SEVA 3.1 or BioBrick vectors, introducing BsaI recognition sites for directional multipart assembly via Golden Gate (Figure 1).

Figure 2.Graphical overview of the SevaBrick Assembly. Inserts and backbones are amplified using a core set of standard primers which introduce compatible Bsal sites, allowing the construction of expression vectors with varying degrees of complexity via Golden Gate (Figure modified from Damalas et al., 2020).

As for all our final composite parts, the initial steps of our cloning strategy constitute the mutagenesis PCR amplification of different genetic elements, followed by the efficient assembly of the PCR amplified genetic products into pSB1C3 backbone. The cloning process is described in detail below:

Step 1

Mutagenesis PCR amplification with Cas13a P1 FWD and Cas13a P1 RVS primers using the pGJK_His-SUMO-LbuCas13a as a template. This PCR produces the P1 part ready for Golden Gate assembly.
Mutagenesis PCR amplification with Cas13a P2 FWD and Cas13a P2 RVS primers using the pGJK_His-SUMO-LbuCas13a as a template. This PCR produces the P2 part ready for Golden Gate assembly.
Mutagenesis PCR amplification with Cas13a P3 FWD and Cas13a P3 RVS primers using the pGJK_His-SUMO-LbuCas13a as a template. This PCR produces the P3 part ready for Golden Gate assembly.
Mutagenesis PCR amplification with Cas13a P4 FWD and Cas13a P4 RVS primers using the pGJK_His-SUMO-LbuCas13a as a template. This PCR produces the P4 part ready for Golden Gate assembly.
PCR amplification with Ev and Pv standard primers from Basic SevaBrick Assembly [seva 3.1] using the Bba_J364007 part of the 2022 DNA distribution Kit. This PCR produced the pSB1C3 backbone linearized and ready for Golden Gate assembly.

Figure 3.1% agarose gel electrophoresis of the PCR amplified SUMO-Cas13a parts and pSB1C3 backbone. DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. Cas13a P1 part: Cas13a P1 part amplified from pGJK_His-SUMO-LbuCas13a with Cas13a P1 FWD and Cas13a P1 RVS primers (1240 bp). Cas13a P2 part: Cas13a P2 part amplified from pGJK_His-SUMO-LbuCas13a with Cas13a P2 FWD and Cas13a P2 RVS primers (734 bp). Cas13a P3 part: Cas13a P3 part amplified from pGJK_His-SUMO-LbuCas13a with Cas13a P2 FWD and Cas13a P3 RVS primers (1338 bp). Cas13a P4 part: Cas13a P4 part amplified from pGJK_His-SUMO-LbuCas13a with Cas13a P4 FWD and Cas13a P4 RVS primers (646 bp). DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. pSB1C3 backbone linearized: pSB1C3 backbone linearized amplified from Bba_J36400 (2022 DNA distribution Kit) with Ev and Pv primers (2052 bp). DNA ladder: DNA ladder 100bp DNA Marker NIPPON Genetics EUROPE

As shown in the above Figure all the PCR amplifications succeeded. Cas13a P1 part (1240 bp), Cas13a P2 part (734 bp) , Cas13a P3 part (1338 bp) , Cas13a P4 part (646 bp) and pSB1C3 backbone linearized (2052 bp) are depicted in the Figure..

Step 2

Golden Gate assembly of the PCR amplified P1, P2, P3, P4 parts with the linearized pSB1C3 vector for the efficient construction of the SUMO-LbuCas13a coding sequence into PSB1C3 plasmid.

To verify the successful Golden Gate assembly, colony PCR was performed using the primers VR and VF2. Then the samples were loaded and run in 1 % agarose gel electrophoresis. As depicted on the following Figure from all colonies the desired SUMO-LbuCas13a (5908 bp) part has been amplified. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20 performing the template DNA of the colony PCR. The other half amount was picked for the overnight liquid culture.

Figure 4.Colony PCR of E-coli DH5a transformants using VR and Cas13a-p4-RVS primers, after Golden Gate assembly. 1 % agarose gel electrophoresis. Lanes 1,2,3,5,6,7,9,10,11: SUMO-LbuCas13a Repos (5908 bp) from different colonies, using VR and Cas13a-p4-RVS primers. DNA ladder: DNA ladder 1kb plus NEB. The bands shown down of the desired band are byproducts because some the primers VR-VF2 enhanced another part of the bacteria.

Since the efficient insertion of the SUMO-LbuCas13a coding sequence into pSB1C3 after multiple mutagenesis PCR and Golden Gate assembly steps, we followed an additional cloning strategy to construct the final SUMO-LbuCas13a coding device under T7 promoter (https://parts.igem.org/Part:BBa_K4170016) necessary for all protein expression experiments. Further information regarding the cloning strategy followed to construct the final device can be found on the SUMO-LbuCas13a coding device under T7 promoter part registry page (BBa_K4170016).

Introduction to protein expression optimization strategies

This part derived from improvement (cis and trans optimization) strategies on the the Cas13a Lbu BBa_K2926001 (https://parts.igem.org/Part:BBa_K2926001) part. This existing part that we used for our experiments and we decided to improve have been designed by iGEM19_Bielefeld-CeBiTec team. This part codes for the Cas13a derived from Leptotrichia buccalis. According to the part’s documentation in the registry the EcoRI and PstI site have been removed to succeed RFC [10] compatibility. This part was used by the Bielefeld-CeBiTec team for assays in S.cerevisiae. The detail cloning strategy to efficiently insert the Cas13a Lbu (BBa_K2926001) under the T7 promoter in place of the SUMO-LbuCas13a "optimized" protein ensuring that all the other plasmid components remain the same, is described on the Contribution (iGEM22_Thessaloniki_Meta) section of Cas13a Lbu (BBa_K2926001) registry page.

Two different optimization approaches were followed for the improvement of the part BBa_K2926001:

Cis-optimization: codon optimization for efficient Cas13a production in ecoli.
Trans-optimization: generate gene chimera for Cas13a production in fusion with SUMO solubility tag.

Cis-optimization: codon optimization for efficient Cas13a production in E.coli.

In the context of synthetic biology, codon optimization is widely used to ameliorate gene expression in heterologous expression systems. Codon optimization is based on the basic principle of the genetic code that the distribution of the 64 unique DNA codons is non-random. Within a genome exist both rare and abundant DNA codons and their distribution varies across all organisms. The host-specific codon usage bias (CUB) influences translation efficiency especially when there is a high dominance of rare codons in the genetic sequence that is intended for translation. A common strategy for common optimization is the replacement of the rare codons with more frequently occurring ones, in accordance with the CUB of the specific organism. Since we decided to express LbuCas13a protein in BL21 (DE3) E.coli strain, we followed a codon optimization approach to achieve maximum protein expression efficiency in the desired E.coli strain.

Trans-optimization: generate gene chimera for Cas13a production in fusion with SUMO solubility tag.

SUMO protein has been previously fused to the N-terminus of several proteins leading to increased expression and solubility of the protein of interest. But how does SUMO protein enhance protein solubility and proper folding? The answer lies in the structure of SUMO protein. Specifically, SUMO has an inner hydrophobic core and an external hydrophilic surface, thus exerts a detergent-like effect in proteins that cannot easily acquire their proper folding. Despite the advantages of SUMO addition, one drawback of this protein expression strategy is the necessary cleavage of the solubility tag. However, many SUMO proteases such as Ulp1, which are members of the cysteine protease superfamily can be used to cleave the SUMO tag without affecting the N-terminus of the desired protein (Panavas et al., 2009). Therefore, by expressing the LbuCas13a protein in fusion with the SUMO-chaperone protein we aim to enhance the quantity of Cas13a protein that accumulates to the soluble cytoplasmic fraction, succeeding proper protein folding and subsequent efficient SUMO tag removal.

Protein expression and comparative experiments with BBa_K2926001 part

To demonstrate that our modifications had a positive effect on the efficiency of Cas13a protein production, we conducted comparative protein production experiments between the “improved” LbuCas13a (https://parts.igem.org/Part:BBa_K4170016) and the analogous protein from BBa_K2926001 part.

Comparative experiments BBa K2926001.png

Cloning strategy of Cas13a Lbu (BBa_K2926001) for comparative experiments.

The CDS of Cas13a Lbu (BBa_K2926001) flanked by appropriate recognition sequences of BsaI restriction enzyme was ordered from IDT and cloned downstream of the T7 promoter in pSB1C3 plasmid with Golden Gate assembly. This part is the Cas13a Lbu part.

The cloning process is described in detail below:

Step 1. PCR amplification

PCR amplification with Ev and Pv standard primers from Basic SevaBrick Assembly (Damalas et al., 2020) using pSB1C3 (Bba_J36400-2022 DNA distribution Kit) a template (Figure 1). This PCR produces the pSB1C3 backbone part ready for Golden Gate assembly.

Figure 5. 1 % agarose gel electrophoresis of the PCR amplified pSB1C3 plasmid with Ev and Pv primers. The estimated DNA bands are 2029bp.

PCR amplification with cas13a T7 P0 FWD and SUMOLESS RVS primers using the LbuCas13a coding device under T7 promoter (BBa_K4170016-link) as a template (Figure 2). This PCR produces the LacI-promoter-RBS part ready for Golden Gate assembly.

Figure 6.1 % agarose gel electrophoresis of the PCR amplified LbuCas13a coding device under T7 promoter (BBa_K4170016-link) with T7 P0 FWD and SUMOLESS RVS primers.

Step 2. Golden Gate-based sevaBrick assembly

Golden Gate assembly of the PCR amplified LacI-promoter-RBS and Cas13a Lbu parts (BBa_K2926001) along with the ‘linearized’ pSB1C3 backbone part for the efficient construction of the LbuCas13a coding sequence under the transcriptional control of the Lac Repressor.

The Golden Gate assembly products underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using the primers VR and VF2. Picking sample from different colonies and then evaluating the results on a 1 % agarose gel electrophoresis we concluded that the Golden Gate assembly was successful in the first colony. The PCR product in the 1st colony has the suitable number of basepairs (5392bp), while the 2nd colony showed no PCR amplification. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked for liquid overnight culture.

Figure 7.1% agarose gel electrophoresis of the PCR amplified product Cas13a Lbu-T7 (BBa K2926001) with VF2 and VR primers

The final plasmid after the Golden Gate assembly contains the same DNA elements/features as the cloned final SUMO-LbuCas13a coding device under T7 promoter (BBa_K4170016), replacing the CDS of the codon-optimized Cas13a with the CDS of Cas13a Lbu (BBa_K2926001). Furthermore, the CDS of the molecular chaperone SUMO sequence has also been removed. In addition, utilizing this cloning strategy we incorporated the 6xHis affinity tag at the N-terminus of the Cas13a Lbu (BBa_K2926001) to facilitate the efficient purification of the protein utilizing a Ni-NTA affinity purification methodology. The genetic sequences of the Cas13a Lbu expression device are illustrated at the following map derived from snapgene. The Cas13a Lbu coding device inserted into the pSB1C3 backbone generated a new composite part which is deposited at the IGEM Registry (BBa_K4170056 https://parts.igem.org/Part:BBa_K4170056 )

Figure 8. Cas13a Lbu expression device in pSB1C3 plasmid backbone

Expression of recombinant LbuCas13a proteins.

General Protein expression workflow

This protocol was followed for all the protein expression experiments with minor modifications at the IPTG concentration, induction temperature or time of induction .

The procedure of the protein expression initiates with the preparation of the bacterial pre-culture (15ml) incubated overnight at 37 °C. The following day the pre-culture was diluted in 1L of nutrient media, followed by a further incubation at 37°C of the diluted pre-culture until Optical Density (OD600) reached 0.7 – 0.8 (continuous measurements at the photometer at specific time points). To induce LbuCas13a protein expression we added IPTG to the 1L bacterial culture to achieve a final concentration of 1mM. The bacterial culture was then incubated for 6 hours at 25°C. After overnight incubation, the bacterial culture was centrifuged at 10.000 g for 20 min at 4 °C and the supernatant was discarded. The bacterial pellets were resuspended in binding buffer (3oomM NaCl, 50mM NaH2PO4, 10mM imidazole, pH 8) followed by successive freeze-thaw cycles. After the freeze-thaw steps, Lysozyme (100mg/ml) and Triton x-100 were added and ultrasonication was performed to lyse the bacterial membrane. After ultrasonication, DNase (1mg/ml), MgCl2 (8mM) and Protease Inhibitor (PI) were added and the samples were incubated in a cold room for 2 hours on a rotator machine. To obtain the soluble fraction of the protein, refrigerated centrifugation was carried out and the supernatant was then filtered by using 0.22 μm filter units. The remaining pellet constitutes the Inclusion Bodies (IBs), which also contains the insoluble form of the protein of interest. To isolate the SUMO-LbuCas13a protein from the IBs, the bacterial pellet is subjected to successive washing steps using 3 different buffers (A, B, C) followed by ultracentrifugation at 30.000g for 20 min at 4 C. The process is completed by resuspending the protein in L-Arginine solution which promotes the proper folding of the protein restoring its functional quaternary structure.

SDS-PAGE gel electrophoresis and Western blotting of Cas13a Lbu (BBa_K2926001)

Equal volumes of protein samples corresponding to the filtered soluble and resuspended insoluble (IBs) solution respectively, were loaded into each well of the SDS-PAGE gel for separation based on molecular weight. After gel electrophoresis completion, suitable images of the gels were obtained. After the separation of the protein mixture through the SDS-PAGE gel electrophoresis, it was transferred to the PVDF membrane for western blotting. After the initial blocking step, the addition of the anti-His primary and the anti-mouse alkaline Phosphatase-Labeled secondary antibody, the protein bands were detected using the alkaline phosphatase detection method.

Figure 9A. 8% SDS-PAGE electrophoresis of Cas13a Lbu (BBa_K2926001) protein after a 6h induction at 25°C using 1mM IPTG. Lane 1: protein marker (nippon genetics, bluestar prestained ), lane 2 : Cas13a soluble part, lane 3 : Cas13a insoluble part (IBs).B. Western blot of Cas13a after a 6h induction at 25°C using 1mM IPTG. Lane 1 : protein marker (nippon genetics blue star prestained), lane 2 : Cas13a soluble part, lane 3 : Cas13a insoluble part (IBs).

Results analysis

Analyzing the results from the SDS-PAGE gel electrophoresis and the Western Blotting (Figure 9A,B) we can conclude that the Cas13a Lbu (BBa_K2926001) protein is detected only in the insoluble fraction which corresponds to the inclusion bodies of the bacteria. The protein band which corresponds to the LbuCas13a protein is detected at the molecular weight of 130kDa. However, no LbuCas13a protein band is detected at the soluble cytoplasmic fraction. The process of obtaining bioactive functional protein from inclusion bodies is usually labor intensive and the yields of the recovered recombinant protein are often low.

SDS-PAGE gel electrophoresis and Western blotting of “improved” SUMO-LbuCas13a protein (BBa_K4170014)

Equal volumes of protein samples corresponding to the filtered soluble and resuspended insoluble (IBs) solution respectively, were loaded into each well of the SDS-PAGE gel for separation based on molecular weight. After gel electrophoresis completion, suitable images of the gels were obtained. After the separation of the protein mixture through the SDS–PAGE gel electrophoresis, it was transferred to the PVDF membrane for western blotting. After the initial blocking step, the addition of the anti-His primary and the anti-mouse alkaline Phosphatase-Labeled secondary antibody, the protein bands were detected using the alkaline phosphatase detection method.

Figure 10 A. 8% SDS-PAGE electrophoresis of SUMO-LbuCas13a (BBa_K4170016) protein after a 6h induction at 25°C using 1mM IPTG. Lane 1: protein marker (nippon genetics, bluestar prestained ), lane 2 : Cas13a soluble part, lane 3 : Cas13a insoluble part (IBs). B. Western blot of Cas13a after a 6h induction at 25°C using 1mM IPTG. Lane 1: protein marker (nippon genetics blue star prestained), lane 2 : Cas13a soluble part, lane 3 : Cas13a insoluble part (IBs).

Results analysis

Analyzing the results from the SDS-PAGE gel electrophoresis and the Western Blotting (Figure 10A ,B) we can conclude that the SUMO-LbuCas13a is detected in both soluble and insoluble fraction (Inclusion Bodies). The protein band which corresponds to the SUMO-LbuCas13a fusion protein is detected at the molecular weight of 155kDa. Therefore, we can assume that the addition of the SUMO solubility tag enhanced the soluble fraction of the LbuCas13a protein. The purification of soluble proteins is less expensive and time consuming compared to the process needed for protein purification and recovery from inclusion bodies. Utilizing the chaperone-mediated LbuCas13a protein recovery from soluble fraction ensures the integrity of the refolded proteins. The resolubilization procedures required to recover the protein from inclusion bodies can disturb the integrity of the protein.

In addition, if we compare the Figures 9 and 10 which correspond to the Cas13a Lbu (BBa_K2926001) and SUMO-LbuCas13a (BBa_K4170014) respectively, we can understand that the codon optimization procedure enhanced the total protein yield in both the soluble and the insoluble fraction of the LbuCas13a protein. Further information about the downstream experiments regarding the SUMO-LbuCas13a purification with His SpinTrap purification columns and the enzymatic removal of the SUMO protein can be found on the results and on the experiments pages of iGEM22_Thessaloniki_Meta team WIKI (https://2022.igem.wiki/thessaloniki-meta/results).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3486
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 349
Illegal AgeI site found at 1564
1000
COMPATIBLE WITH RFC[1000]

Citations

1.Butt, T., Edavettal, S., Hall, J. and Mattern, M., 2005. SUMO fusion technology for difficult-to-express proteins. Protein Expression and Purification, 43(1), pp.1-9.

2.Damalas, S., Batianis, C., Martin‐Pascual, M., Lorenzo, V. and Martins dos Santos, V., 2020. SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial Biotechnology, 13(6), pp.1793-1806.

3. Lipońska, A., Ousalem, F., Aalberts, D., Hunt, J. and Boël, G., 2018. The new strategies to overcome challenges in protein production in bacteria. Microbial Biotechnology, 12(1), pp.44-47.