Part:BBa_K2643001

dxCas9 (HIS Tag)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3823
  Illegal BglII site found at 251
  Illegal BglII site found at 1188
  Illegal BamHI site found at 2006
  Illegal XhoI site found at 3544
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  Illegal NgoMIV site found at 4139
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2886
  Illegal BsaI site found at 3740
  Illegal BsaI.rc site found at 1411
  

Usage and Biology

The xCas9 variant is derived from the common Streptococcus pyogenes Cas9 (SpCas9). SpCas9 is part of the CRISPR (clustered regularly interspaced short palindromic repeat) Cas system . This system is an adaptive microbial defense system protecting the cells from invading phages and other harmful mobile genetic elements. The mechanism of SpCas9 can be programmed to recognize and cleave virtually any sequence preceding a 5′-NGG-3′ Protospacer Adjacent Motif (PAM) sequence via its RNA-guided endonuclease . The xCas9 has evolved from SpCas9 and includes point mutations in amino acids E480K, E543D, E1219V, A262T, S409I and M694I that allow the recognition of a broader PAM sequence including NG, GAA and GAT . The xCas9 protein was obtained by phage assisted continuous evolution . By mutating the endonuclease activity of xCas9, the cleavage inactive version of xCas9 was obtained and called dxCas9. This specific biobrick is the catalytically dead version of xCas9, lacking endonuclease activity (dxCas9).

The dxCas9 coding sequence was cloned into the pACYCDuet-1 expression vector flanked by T7 promoter and terminator and transformed into Escherichia coli BL21AI for its expression. Induction with IPTG and arabinose allowed stable expression of this protein for its purification and characterization in vitro. The dxCas9 from this biobrick can be used in several applications where normal dCas9 is used, but with the advantage of higher versatility and specificity of the recognition activity (Hu, et al., 2018). This biobrick was tested by the iGEM TU Delft team (2018) as a DNA binding protein. Additionally, the dxCas9 was fused to Tn5 transposase (BBa_K2643002) with a linker (BBa_K2643003) to create a fusion protein (BBa_K2643000).

Characterization

Introduction

In order to fully characterize the dxCas9 biobrick, it had to be constructed, expressed, purified and tested for its in vitro functionality. The dxCas9 coding sequence was cloned into the pACYCDuet-1 with a T7 promoter and a N-terminal His-tag, and this plasmid was transformed into BL21 AI for protein expression. The dxCas9 protein was purified using both nickel affinity chromatography and heparin chromatography Finally, in vitro functionality was tested with two assays: a trypsin resistance assay to check for proper sgRNA loading of the dxCas9, and an electrophoretic mobility shift (binding) assay (EMSA) to check for proper complex formation with target DNA.

Strain construction

Aim

Construct two plasmids harbouring the dxCas9 coding sequence, namely pACYCDuet-1 for expression and pSB1C3 for iGEM biobrick submission.

Part A: Construction of dxCas9 in pACYCDuet-1 procedure

The construction of pACYCDuet-1_dxCas9 is performed by restriction ligation cloning of dxCas9 into the expression vector pACYCDuet-1 under the T7 promoter and terminator.

Plasmid #108383 from Addgene containing dxCas9 (3.7) from Dr. David Liu was used as template for dxCas9 amplification. Primers forward (5’-atatagcggccgctatggacaagaagtactccat-3’) and reverse (5’-tatggtaccttatggccggcccaccttcctcttcttcttgg-3’) were used for PCR amplification of dxCas9 from the template dxCas(3.7)-VPR. The primers include 5’ overhangs containing respectively the NotI and KpnI restriction enzyme sites in the forward and reverse primer respectively.

pACYCDuet-1 (Novagen) is designed for coexpression of two genes. The plasmid contains two multiple cloning sites (MCS), each of which is preceded by a T7 promoter/lac operator and ribosome binding site (rbs). The P15A replicon is present to sustain plasmid replication and the chloramphenicol resistance gene allows for applying selective pressure to cells for maintaining the plasmid.

The vector pACYCDuet-1 was isolated from the E.coli DH5α cells according to the [http://2018.igem.org/Team:TUDelft/Experiments#plasmidisolation-scroll plasmid isolation protocol]. Then, the dxCas9 PCR amplicon was restricted with enzymes NotI-HF and KpnI-HF (New England Biolabs) as well as the pACYCDuet-1 plasmid, according to the [http://2018.igem.org/Team:TUDelft/Experiments#restriction-scroll restriction protocol]. The pACYCDuet-1 vector and dxCas9 insert were ligated using T4 DNA ligase and subsequently the ligated product was transformed into chemical competent E. coli DH5α cells using the [http://2018.igem.org/Team:TUDelft/Experiments#chemcomptcellstrans-scroll chemical competent cell transformation protocol]. Notably, by using the combination of NotI and KpnI restriction enzymes, one of the T7 promoters is excised, which is in this case not a problem.

Transformed cells were screened via colony PCR using forward primer T7 promoter (5’-taatacgactcactataggg-3’) and reverse primer T7 terminator (5’-gctagttattgctcagcgg-3’) that amplify the sequence between the promoter and terminator. This way, fragments cloned into the multiple cloning site (located between the T7 promoter and terminator) of pACYCDuet-1 will be amplified.

Results

The colony PCR identified two positive colonies with possibly correct integration of dxCas9 in pACYCDuet-1 (Figure 1). Colony 3 (figure 1, lane 4) was grown overnight in liquid media for plasmid extraction. The insert in the purified plasmid was sequence verified with six sets of primers: T7 promoter (5’-taatacgactcactataggg-3’), T7 terminator (5’-gctagttattgctcagcgg-3’), FP003 (5’-gcgaattttccaaaagagtgatcc-3’), FP012 (5’-ggaatactgcaaaccgttaagg-3’), FP013 (5’-ccgttttgcaggtagtacag-3’) and P514 (5’-5'-GATGGTGTCCGGGATCTC-3’). Glycerol stocks of these cells were stored at -80 ºC and plasmid isolated for further characterization of the biobrick.

Figure 1. Colony PCR of dxCas9, expected size 4149bp. The ladder represents the size of DNA in bps.

Part B: Construction of dxCas9 in pSB1C3 procedure

Plasmid BBa_J04450 from the iGEM registry was used as template for obtaining the linear pSB1C3 iGEM BioBrick Backbone. This BioBrick contains a mRFP expression cassette that turns cells harbouring the plasmid red. BBa_J04450 was transformed into chemically competent E.coli DH5α cells. Red colonies were grown in LB + chloramphenicol and the plasmid was isolated. The plasmid BBa_J04450 was used to obtain the iGEM BioBrick backbone pSB1C3 by PCR using forward primer (5’-TACTAGTAGCGGCCGCTGC-3’) and reverse primer (5’-CTAGAAGCGGCCGCGA-3’). The primers amplify the iGEM pSB1C3 BioBrick backbone from the suffix until the prefix.

The dxCas9 was amplified from the previously constructed plasmid pACYCDuet-1_dxCas9 by PCR using forward primer (5’- TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGatggacaagaagtactccattgg-3’) and reverse primer (5’- CTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTAttatggccggcccac-3’).

The primers contain a 5’ overhang for Gibson assembly of the dxCas9 into the pSB1C3 iGEM Biobrick backbone.

The pSB1C3 backbone and dxCas9 were combined by Gibson assembly. Subsequently, the Gibson assembly product was transformed into chemical competent E. coli DH5α cells using heat shock [http://2018.igem.org/Team:TUDelft/Experiments#chemcomptcellstrans-scroll chemical competent cell transformation protocol].. The transformation resulted in white colonies, indicating that an insert is integrated, other than mRFP. The parental plasmid is not present anymore.

Results

Twenty white colonies were screened via colony PCR using forward primer VF2 (5’-tgccacctgacgtctaagaa-3’) and reverse primer VR (5’-attaccgcctttgagtgagc-3’) that amplify the DNA from before the prefix until after the suffix. This way, any fragment cloned between the prefix and suffix would be amplified.

The colony PCR resulted in six colonies with the potentially correct integration of dxCas9 in pSB1C3. Three positive colonies, colony 1, 7 and 15 (figure 2, lane 2, 8, 17), were grown overnight in liquid media supplemented with chloramphenicol. After plasmid isolation, colony number 15 (figure 2, lane 17) was sequenced verified. The primers used for sequence verification can be found in table 1.

Figure 2. Colony PCR of dxCas9, expected size 4461bp. 0.8% Agarose. The ladder represents the size of DNA in bps.

Table 1. Primers for sequence verification dxCas9 in pSB1C3.

Glycerol stocks of the cells harbouring the pSB1C3_dxcas9 plasmid were stored at -80ºC and the plasmid was sent to the iGEM registry.  

Upstream processing: Expression

Aim

Transform the plasmid into the expression host (E.coli BL21 AI) and express the dxCas9 protein.

Cultivation Procedure

The dxCas9 was expressed according to the [http://2018.igem.org/Team:TUDelft/Experiments#proteinexpression-scroll protein expression protocol] on our wiki. To summarize, LB media was inoculated with an overnight culture at OD600 ~0.02 and grown to OD600 ~0.5 in 5L shake flasks at 37 °C with 180 rpm. At this point, the cells were placed on ice for 30 min and induced with 1mM IPTG and 0.2% arabinose. The cells were then incubated for 16 hours at 18 °C and 180 rpm, for production of the dxCas9 protein. After expression, the cells were harvested and washed with PBS using centrifugation at 5200 xg for 15 minutes and 4 °C.

Results

A 12% SDS PAGE was run to analyse the expression (figure 3). A significant amount of dxCas9 is observed in the crude lysate (figure 3, lane 3), which is not present in the pre-induced sample (figure 3, lane 2). This suggest that there is no leaky expression, and that the cultivation went as expected.

Figure 3. 12% Tris-Glycine SDS PAGE of the expression of dxCas9. The ladder represents the molecular weight in kDa.

Downstream processing: Purification

Aim

Purify the dxCas9 from the cells that were received from upstream processing.

Purification Procedure

The dxCas9 protein was purified according to the [http://2018.igem.org/Team:TUDelft/Experiments#proteinpurificationdxCas9-scroll dxCas9 protein purification protocol] on our wiki. To summarize, the cells were lysed with a high pressure homogenizer (2 rounds at 1 kbar) in lysis buffer (20 mM Tris-HCL, 250 mM NaCl, 1 mM DTT, 5 mM imidazole, protease inhibitor, pH 8.0). After clarifying the lysate via centrifugation for 45 min at 16,000 xg, the dxCas9 protein was purified by Nickel affinity gravity column chromatography . The chromatography steps are described in table 2.

Table 2. Nickel affinity Chromatography protocol.

All fractions were analysed with SDS-PAGE, and fractions containing the dxCas9 protein were pooled and further purified using heparin chromatography , 2017; on the AKTA pure with a 1mL HiTrap Heparin HP column.The chromatography steps are described in table 3:

Table 3. Heparin Chromatography protocol.

All fractions were analysed with SDS-PAGE, and fractions containing the dxCas9 protein were pooled and dialysed twice against 1L of dialysis buffer (20 mM Tris-HCl, 100 mM KCl, 5% v/v glycerol, 1 mM beta-mercaptoethanol and pH 7.5) for an hour using a Pierce 10 kDa molecular weight cut off cassette. Post dialysis the samples were frozen at -20℃ and stored for functionality testing assays.

Results

For each purification step the protein can be tracked with 12% tris-glycine [http://2018.igem.org/Team:TUDelft/Experiments#SDSPAGEelectrophoresis-scroll SDS-PAGE analysis] according to the protocol on our wiki. Included in the gel (Figure 4, 6, and 7) are samples collected during cell lysis, Nickel affinity chromatography, Heparin chromatography, and the final dxCas9 protein after dialysis.

The nickel chromatography was not completely successful, but improved significantly after an initial optimization of the concentration of imidazole in the binding buffer. For concentrations higher than 5mM imidazole, the dxCas9 did not bind to the nickel resin and the majority of the dxCas9 protein was observed in the flowthrough and wash. It is recommended to further optimize the nickel chromatography to further reduce losses (figure 4, lane 6-8), and reduce contaminating proteins due to non-specific binding to the nickel resin (figure 4, lane 9-10). However for our purposes, the losses were tolerated, and only elution fractions 3 to 6 were pooled (figure 4, lane 11-14).

Figure 4. 12% Tris-Glycine SDS PAGE of the nickel chromatography of dxCas9. The ladder represents the molecular weight in kDa.

Additionally two bands for the dxCas9 were observed after nickel chromatography. One at the expected weight, and one slightly higher. It was unclear why the dxCas9 would run higher. As a following step, elution fraction 3 (figure 4, lane 11) was run on a 6% SDS PAGE gel under different denaturing conditions for better resolution (Figure 5).

Figure 5. 6% Tris-Glycine SDS PAGE of the elution fraction 3 from nickel chromatography of dxCas9 under different denaturing conditions. The ladder represents the molecular weight in kDa.

On the 6% tris-glycine SDS PAGE a single band for dxCas9 was observed for all conditions, suggesting that the double band might be some artifact of the 12% SDS PAGE. To further confirm these results, both bands from the 12% gel were excised and sent for mass spectrometry. The mass spectrometry confirmed that both bands were full length dxCas9 with both the N and C terminal intact. This concluded that there was not any protease digestion and that the results are potentially due to a gel artifact. As a result, the purification was continued.

The heparin chromatography was successful. The recoveries were high, with no losses in the flowthrough and wash (figure 6, lane 3-4). The first 4 elution fractions (figure 6, lane 5-8) contained some low molecular weight contaminating proteins, as a result only elution fraction 5 to 10 (figure 6, lane 9 to 14) were pooled. Again the double band for dxCas9 was observed on the 12% SDS PAGE.

Figure 6. 12% Tris-Glycine SDS PAGE of heparin chromatography of dxCas9. The ladder represents the molecular weight in kDa.

After dialysis, the sample was run on a 8% SDS PAGE (figure 7). Note the dxCas9 is only one band. Trace amounts of contaminating background is observed, however dxCas9 is significantly more in quantity and the background was tolerated for functionality testing. The final concentration of the solution was determined according to the [http://2018.igem.org/Team:TUDelft/Experiments#BCAproteinquantification-scroll BCA protocol] on our wiki and resulted in a concentration of 0.46mg/mL or 2.88µM.

Figure 7. 8% Tris-Glycine SDS PAGE of the final dxCas9 post dialysis. The ladder represents the molecular weight in kDa.

In vitro functionality testing: Trypsin (sgRNA binding) assay

Aim

To confirm that the dxCas9 can be loaded with sgRNA by performing a trypsin resistance test.

Assay Procedure

The in vitro functionality was tested according to the [http://2018.igem.org/Team:TUDelft/Experiments#dxCas9trypsinresistance-scroll Trypsin resistance (sgRNA binding) assay protocol] on our wiki. To summarize, 1.152µM purified dxCas9 was loaded with 1.152µM sgRNA provided by Arbor Biotechnologies for 10 minutes at 37°C. Trypsin was added in a 1:10, 1:7, and 1:5 Trypsin:dxCas9 molar ratio to the sgRNA:dxCas9 complex and incubated at 23°C for 30 minutes. All DNA and RNA sequences can be found in table 4.

Table 4. Spacer sequences of all sgRNA (BBa_K2643012) and EPO (target DNA BBa_K2643004) used in this study.

Results

When the protein is loaded with sgRNA it has been proven that the dxCas9 undergoes a significant conformational shift causing the protein to become significantly more trypsin (protease) resistant compared to an apo-dxCas9 protein . When comparing the intact dxCas9 band (158.3kDa) and the degradation pattern (bands <158.3kDa) of the sgRNA loaded dxCas9 proteins (Figure 8A, lane 5-15 and Figure 8B, lane 2-13) to the apo-dxCas9 protein (Figure 8A, lane 2-4 and Figure 8B, lane 14-15), it’s clear that loaded dxCas9 proteins have higher trypsin resistance as literature suggests.These results also indicate that certain sgRNAs have stronger binding to the dxCas9. sgRNA 2.1 (Figure 8A, lane 14-15), 2.2 (Figure 8A, lane 11-13), and 3.2 (Figure 8B, lane 8-10) have stronger binding to the dxCas9 compared to the other sgRNA, since dxCas9 loaded with these sgRNAs has higher trypsin resistance than the others. A similar assay can be executed to test the binding affinity of the sgRNA array.

Figure 8A. 12% Tris-Glycine SDS PAGE of the trypsin resistance assay for gRNA J1.1, J1.2, J2.1, and J2.2. The ladder represents the molecular weight in kDa. Figure 8B. 12% Tris-Glycine SDS PAGE of the trypsin resistance assay for gRNA J2.3, J3.1, J3.2, and J4.1. The ladder represents the molecular weight in kDa.

In vitro functionality testing: Electrophoretic mobility shift (binding) assay (EMSA)

Aim

To confirm that the sgRNA loaded dxCas9 can form a complex with the target DNA (in this case, EPO CDS).

Procedure

The in vitro functionality was tested according to the electrophoretic [http://2018.igem.org/Team:TUDelft/Experiments#dxCas9mobilityshift-scroll mobility shift (binding) protocol] on our wiki. To summarize, 0-1152nM purified dxCas9 were loaded with 0-1152nM sgRNA provided by Arbor Biotechnologies in reaction buffer (20 mM Tris-HCl, 100 mM KCl, 5% v/v glycerol, 1 mM beta-mercaptoethanol, 10mM MgCl2, with and without 0.1mM EDTA, pH 7.5 ) for 10 minutes at 37°C. 25nM Target DNA were added to the sgRNA:dxCas9 complex and incubated at 37°C for 1 hour. Two negative controls were also performed, one without sgRNA and one with an off-target DNA (kanamycin CDS). All DNA and RNA sequences can be found in table 5.

Table 5. The sequence for sgRNA (BBa_K2643012), EPO (target DNA BBa_K2643004) and KAN (off target DNA BBa_K2643011).

Results

A mobility shift occurs when binding of DNA to protein retards the movement of the DNA through the polyacrylamide gel, creating two distinct bands. A mobility shift in the target DNA was observed for 576-1152nM dxCas9 loaded with sgRNA (figure 9, lane 4-5), however mobility shifts are not observed when dxCas9 is incubated without sgRNA (figure 9, lane 6-9). Furthermore, no mobility shifts were observed when dxCas9 was incubated with off target DNA (figure 9, lane 10-12) (DNA that does not have a complementary sequence to the sgRNA). These results indicate that the sgRNA-loaded-dxCas9 can form a complex with only target DNA which has a complementary sequence to the sgRNA. The lower band at 632bp is the target DNA alone (figure 9, lane 1-9), and the higher band at >1000bp is the target DNA in the dxCas9 complex (figure 9, lane 4-5).

Figure 9. Target DNA mobility shift with negative controls as observed on 5% TBE native PAGE. The ladder represents the size of DNA in bps.

Summary

The dxCas9 biobrick was fully characterized, from strain construction to in vitro functionality testing. The in vitro functionality testing proved that the dxCas9 is functional. The trypsin resistance assay proved the dxCas9 was loaded with sgRNA. The EMSA proved that the dxCas9 could form a complex with the target DNA.

Source

Plasmid #108383 from Addgene containing dxCas9 (3.7) from Dr. David Liu was used as template for dxCas9 amplification.

Safety

This biobrick contains a CRISPR protein. The project establishes a sequencing tool using CRISPR-Cas technology. We used a catalytically inactive variant of Cas9, called dxCas9. This means the machinery is incapable of inducing double strand breaks in a target sequence. Therefore, all of the strains created in this project are lacking gene drive possibilities.