Part:BBa_K2643000
dxCas9-linker-Tn5 fusion protein (HIS Tag)
dxCas9-Linker-Tn5
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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
Illegal NgoMIV site found at 5582
Illegal NgoMIV site found at 5607 - 1000INCOMPATIBLE 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
This fusion protein consists of a catalytically inactive version of xCas9, called death xCas9 (dxCas9, BBa_K2643001), fused to a hyperactive transposase (Tn5, BBa_K2643002) connected via a short linker (BBa_K2643003) of 18 amino acids (K L G G G A P A V G G G P K A A D K) in between. The dxCas9 domain of the fusion protein binds to a specific DNA sequence based on the complementary matching between target DNA and sgRNA. Moreover, the target sequence has to present a Protospacer Adjacent Motif (PAM) of either NGN, GAA or GAT [1] in order to have proper guidance of the fusion protein to the target DNA. The Tn5 domain can add a specific sequence of DNA (also called transposon) at a non specific double strand DNA sequence [2]. This fusion is built in a way to make Tn5 integrate the transposon next the the target sequence recognized by dxCas9. Since Tn5 forms dimer during transposon loading process, our fusion protein will dimerize, consisting two Tn5 molecules and two dxCas9 molecules.
As this is a fusion of two different proteins, the source of each protein is also different. The Tn5 transposase is a hyperactive version of Tn5 from Escherichia coli [3] whereas the dxCas9 is a derivative from common Cas9 from Streptococcus pyogenes generated by Hu, et al. [1]. The xCas9 mutant has point mutations in the active site that allow the recognition of a broader Protospacer Adjacent Motif (PAM) sequence, while dCas9 is the dead version of Cas9, lacking endonuclease activity.
The fusion was cloned into pACYCDuet-1 expression vector flanked by T7 promoter and terminator and transformed into Escherichia coli BL21 AI for its expression. Induction with 1mM IPTG and 0.2% arabinose (see below for more details) allowed stable expression of this protein for its purification and characterization in vitro. The fusion from this biobrick is intended to be used in a targeted next generation sequencing platform as a specifically guided tagmentation agent. For such application, the ‘transposon’ sequence that is recognized by Tn5 has been split into two separate molecules (adapters) suitable for the next generation sequencing platform from Oxford Nanopore Technologies. This protein has been characterized to guide the integration of specific adapters to a prefered sequence guided by dxCas9. This activity has been characterized and proven by in vitro assays described in detail below.
The fusion protein was screened for its activity as an in vivo genetic modification tool to specifically introduce DNA sequences. The assays included the introduction of an antibiotic resistance gene guided to LacZ gene in the bacterial genome to generate a knockout mutant. This assay was not successful hence the protein has not shown in vivo activity under the conditions tested. For more detailed information on this characterization, go to BBa_K2643011.
Characterization
Introduction
In order to fully characterize the dxCas9-Linker-Tn5 fusion protein, it had to be designed, constructed, expressed, purified and tested for its in vitro functionality. The dxCas9-Linker-Tn5 coding sequence was cloned into the pACYCDuet-1 expression vector with a T7 promoter and a His-tag. The resulting plasmid was transformed into E. coli BL21 AI for protein expression. The fusion protein was purified using both heparin chromatography [4];[5] and MonoQ chromatography, removing the majority of the contaminants. Finally, in vitro functionality of the fusion protein was tested by developing and utilizing two assays. The first assay, a electrophoretic mobility shift (binding) assay (EMSA), focused on the functionality of the individual components, the Tn5 and dxCas9, of the fusion separately. The assay was used to check for proper complex formation between the fusion protein and the adapter DNA [3]. Additionally, the EMSA was used to validate binding of the fusion protein to the target DNA based on the sgRNA target sequence [6]. The second assay, a targeted integration assay, focused on the functionality of both components together. The integration assay was used to analyse the fusion protein for targeted integration of the adapters close to the target site to which the dxCas9 is guided to by the sgRNA [6];[7].
Strain construction
Aim
Construct two plasmids harbouring dxCas9-Linker-Tn5 coding sequence: in pACYCDuet-1 for expression and in pSB1C3 for iGEM biobrick submission.
Construction of dxCas9-lin-Tn5 in pACYCDuet-1 procedure
To construct the fusion protein, initially Linker-Tn5 DNA was cloned into pACYCDuet-1, for the later integration of dxCas9 in the 5’ site of the linker.
Part A: Linker-Tn5 in pACYCDuet-1 procedure
The construction of Tn5 is performed by restriction ligation cloning of Tn5 into the expression vector pACYCDuet-1 under the T7 promoter and terminator.
Plasmid #60240 from Addgene, pTXB1-Tn5 from Picelli et al. [3], was used as template for Tn5 amplification. In this amplification, the linker sequence that will be located between the dxCas9 and Tn5 sequences is implemented. In order to extend the Tn5 with linker, one of the primers is extended at the 5’ end with the linker sequence. Forward primer (5’-tataggccggcctaaactgggcggcggcgcgccggcggtgggcggcggcccgaaagcggcggataaaatgattaccagtgcactgcatc-3’) and reverse primer (5’-tataGGTACCttagattttaatgccctgcgcca-3’) were used for PCR amplification of Tn5 from the template pTXB1-Tn5 simultaneously integrating the linker. The primers also include 5’ overhangs containing respectively the FseI 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 Linker-Tn5 PCR amplicon was restricted with enzymes FseI-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 Linker-Tn5 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].
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. Additionally, the transformed cells were screened for presence of the Tn5 coding sequence using forward primer Tn5 (5’-tataGAATTCtatgattaccagtgcactgcatc-3’) and reverse primer (5’-tataGGTACCttagattttaatgccctgcgcca-3’).
Results
The colony PCR identified three positive colonies with possibly correct integration of Tn5 in pACYCDuet-1 (Figure 1, lane 8, 9 and 10), resulting in the pACYCDuet-1_Lin-Tn5 plasmid.
Transformant 9 was grown overnight in liquid media and its plasmid was subsequently isolated, purified and sequence verified. Glycerol stocks of the transformant were stored at -80 ºC and its plasmid isolated for further construction of the biobrick.
Part B: dxCas9 into linker-Tn5 pACYCDuet-1 procedure
The construction of pACYCDuet-1_dxCas9-Lin-Tn5 is performed by restriction ligation cloning of dxCas9 into the expression vector pACYCDuet-1_Lin-Tn5 under the T7 promoter and terminator.
Plasmid #108383 from Addgene containing dxCas9 (3.7) from Dr. David Liu [1] 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 FseI restriction enzyme sites in the forward and reverse primer respectively.
The vector pACYCDuet-1_Lin-Tn5 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 FseI-HF (New England Biolabs) as well as the pACYCDuet-1-Lin-Tn5 plasmid, according to the [http://2018.igem.org/Team:TUDelft/Experiments#restriction-scroll restriction protocol]. The pACYCDuet-1_Lin-Tn5 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 FseI 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. Additionally, transformed cells were screened for presence of dxCas9 and Tn5 separately. For this colony PCR forward primer (5’-atatagcggccgctatggacaagaagtactccat-3’) and reverse primer (5’-tatggtaccttatggccggcccaccttcctcttcttcttgg-3’) were used to amplify dxCas9 and in separate colony PCR reaction with forward primer (5’-tataGAATTCtatgattaccagtgcactgcatc-3’) and reverse primer (5’-tataggtaccttagattttaatgccctgcgcca-3`) to amplify the Tn5.
Results
Colony PCR resulted in several colonies with potentially correct integration of dxCas9 into pACYCDuet-1_Lin-Tn5 (figure 2, 3 and figure 4), resulting in the plasmid pACYCDuet-1_dxCas9_Lin-Tn5. Figure 3 verifies that all colonies except for colonies 1, 10 and 11 harbour dxCas9. Figure 4 shows that all the colonies harbour Linker-Tn5 DNA in the pACYCDuet-1 plasmid.
Transformant 4 was grown overnight in liquid media and its plasmid was isolated, purified and sequence verified. Glycerol stocks of these cells were stored at -80 ºC and more plasmid was isolated for further characterization of the biobrick.
Construction of dxCas9-lin-Tn5 in pSB1C3 procedure
The construction of dxCas9-linker-Tn5 in the iGEM compatible pSB1C3 BioBrick backbone is performed by Gibson assembly cloning of dxCas9-Lin-Tn5 into the iGEM backbone.
Plasmid BBa_J04450 from the iGEM registry was used as template for obtaining the linear pSB1C3 iGEM BioBrick Backbone. This BioBrick contains mRFP expression cassette, turning the cell harbouring the plasmid from a white into a red color. First, BBa_J04450 was transformed into E. coli DH5α cells after which the red colonies were inoculated in LB chloramphenicol medium and the plasmid was isolated. In a PCR amplification, the iGEM BioBrick backbone pSB1C3 was obtained with using forward primer (5’-TACTAGTAGCGGCCGCTGC-3’) and reverse primer (5’-CTAGAAGCGGCCGCGA-3’) on the BBa_J04450 DNA template according to the [http://2018.igem.org/Team:TUDelft/Experiments#colonyPCR-scroll colony PCR protocol]. The primers amplify the iGEM pSB1C3 BioBrick backbone from the suffix until the prefix.
The previously constructed plasmid pACYCDuet-1_dxCas9-Lin-Tn5 was inoculated and 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-Lin-Tn5 coding sequence was PCR amplified using forward primer (5’- TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGatggacaagaagtactccattgg-3’) and reverse primer (5’- CTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTAttagattttaatgccctgcgccatc-3’).
The primers included 5’ overhangs compatible for Gibson Assembly with the pSB1C3 backbone. These overhangs would be flanking the dxCas9-linker-Tn5 and are used for cloning purposes of dxcas9-linker-Tn5 into the pSB1C3 iGEM Biobrick backbone.
The pSB1C3 backbone and dxCas9-linker-Tn5 were combined with the use of Gibson assembly. Subsequently, the Gibson assembly product was transformed into chemical competent E.coli DH5α cells using heat shock via the [http://2018.igem.org/Team:TUDelft/Experiments#chemcomptcellsprep-scroll Chemical competent cell transformation protocol]. The transformation resulted in white colonies.
Twenty white colored transformed cells were screened via colony PCR using forward primer VF2 (5’-tgccacctgacgtctaagaa-3’) and reverse primer VR (5’-attaccgcctttgagtgagc-3’) that amplify the DNA from the before the prefix until after the suffix. This way, any fragment cloned between the prefix and suffix would be amplified.
Results
The colony PCR resulted in eight possible colonies (Colony number 1, 2, 8, 9, 12, 16, 19, 20. Figure 5, lane 2, 3, 9, 10, 13, 18, 21, 22) with correct integration of dxCas9-linker-Tn5 in pSB1C3.
Three positive colonies, colony 1, 2 and 8, were grown overnight in liquid media supplemented with chloramphenicol and after [http://2018.igem.org/Team:TUDelft/Experiments#plasmidisolation-scroll plasmid isolation], colony number 8 was sent for sequencing and insertion of dxCas9-linker-Tn5 in pSB1C3 was verified. The primers used for sequence verification can be found in table 1.
Table 1. Primers for sequence verification dxCas9-linker-Tn5 in pSB1C3.
Glycerol stocks of the cells harboring the pSB1C3_dxCas9-linker-Tn5 plasmid were stored at -80 ºC 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 fusion protein.
Cultivation procedure
The fusion protein was expressed according to the [http://2018.igem.org/Team:TUDelft/Experiments#proteinexpression-scroll protein expression protocol] on our wiki. In summary, 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 rotation. 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 fusion protein. After expression, the cells were harvested and washed with PBS using centrifugation at 5200 xg for 15 minutes and 4 °C.
Results
A 8% SDS PAGE was run to analyze the expression. Fusion is observed in the crude lysate (figure 6, lane 2).
Downstream processing: Purification
Aim
Purify the fusion from the cells that were received from upstream processing.
Purification procedure
The fusion was purified according to the [http://2018.igem.org/Team:TUDelft/Experiments#proteinpurificationTn5dxCas9-scroll Fusion 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, 1mM PMSF, pH 7.5). After clarifying the lysate via centrifugation for 45 min at 16,000 xg, the fusion protein was filtered through a 0.45µm filter and then purified by heparin chromatography on the AKTA pure with a 1mL HiTrap Heparin HP column. Note the fusion does have a His-tag, but nickel affinity chromatography was not successful, and the fusion did not bind the to the nickel bead. The chromatography steps are described in table 2:
Table 2. Heparin Chromatography protocol.
All fractions are analyzed with SDS-PAGE, and fractions containing the fusion protein are pooled and further purified using MonoQ chromatography on the AKTA pure with a 1mL MonoQ 5/50 GL column. The chromatography steps are described in table 3:
Table 3. MonoQ Chromatography protocol.
All fractions were analyzed with SDS-PAGE, and fractions containing the fusion 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 fusion was concentrated 6x with an Amicon Ultra-15 with a 10 kDa molecular weight cut off and then frozen at -20℃ and stored for functionality testing assays.
Results
For each purification step, the protein can be tracked with 8% tris-glycine [http://2018.igem.org/Team:TUDelft/Experiments#SDSPAGEelectrophoresis-scroll SDS-PAGE analysis] according to the protocol on our wiki. Included are samples collected during cell lysis, Heparin chromatography, MonoQ chromatography, and the final concentration of the protein (figure 7, 8, and 9).
Degradation was suspected for our fusion protein. Results seem to suggest that the fusion protein is being cleaved at the linker, resulting in both the dxCas9 and the fusion protein being present in our sample. The heparin chromatography was relatively successful at separating the two populations. The dxCas9 has a lower affinity to the heparin resin as a result it elutes at lower concentrations of salt (figure 7, lane 2-10). As the concentration of salt increases less dxCas9 and more fusion elutes (figure 7, lane 6-10). At high levels of salt the mostly fusion elutes (figure 7, lane 11-13). Only these last three elution fractions are pooled (figure 7, lane 11-13). Thus losses are tolerated, to obtain a relatively pure fusion fraction which can be further purified by MonoQ.
The MonoQ chromatography was used as the last purification. The elution peak is broad (figure 8, lane 6-14), however, the fractions are relatively pure and the dxCas9 contamination was mostly eliminated.
After concentration, the sample was run on an 8% SDS PAGE (figure 9). Trace amounts of contaminating background was observed, however, the fusion was 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] and resulted in a concentration of 0.57mg/mL (2.64µM).
In vitro functionality testing: DNA binding assay
Aim
Confirm the binding function of each portion, the Tn5 and the dxCas9, of the fusion separately. Confirm the ability of Tn5 of the fusion protein to load the adapter DNA containing the mosaic ends (ME) sequence[3]. Confirm the ability of dxCas9 of the fusion protein to load sgRNA and bind to the target DNA [6]. When the loaded protein binds to DNA, a mobility shift of the Cy5 labeled adapter or Cy5 labeled target DNA can be observed on a 5% TBE native PAGE. The two different EMSA were performed separately.
Procedure
Part A: Fusion loading with adaptors DNA
The in vitro functionality was tested according to the adaptor DNA [http://2018.igem.org/Team:TUDelft/Experiments#tn5fusionmobilityassay-scroll electrophoretic mobility shift protocol] on our wiki. To summarize this method, 2.5µM Cy5 labeled adapter was incubated with ~0 - 2.5µM fusion protein in reaction buffer (20 mM Tris-HCl, 100 mM KCl, 5% v/v glycerol, 1 mM beta-mercaptoethanol, 0.1mM EDTA, pH 7.5) for 1 hour at 23°C to test the loading of adapter DNA to the fusion protein [7].
Part B: Fusion loading with target DNA
The in vitro functionality was tested according to the [http://2018.igem.org/Team:TUDelft/Experiments#dxCas9mobilityshift-scroll target DNA electrophoretic mobility shift protocol] on our wiki. To summarize this method, 0 - 804 nM purified fusion protein was incubated with 0 - 1340 nM 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. Then 1.22nM Cy5 labeled target DNA was added to the sgRNA:fusion complex and incubate at 37°C for 1 hour. A negative control without sgRNA was also performed. [4];[5]. Table 4 displays the sequences for all reagents.
Table 4. The sequence for adapter, sgRNA (BBa_K2643013) , EPO (target DNA BBa_K2643004). Note that the 5’ of the adapter upper strand is phosphorylated and the 5’ of the adapter lower strand is cy5-labelled.
Please note: The two reactions were incubated separately and were subsequently ran separately on seperate TBE native PAGE.
Results
Part A: Adapter DNA mobility shift
A mobility shift of the adapter DNA is observed for >1.5µM fusion protein (figure 10, lane 7-9). This indicated the ability of the Tn5 portion of the fusion protein to pick up the adapter DNA. This complex retards the movement of the DNA through the polyacrylamide gel, creating the two distinct bands indicated by the arrows. The lower bands around the 50 bp is the unloaded adapter DNA (figure 10, lane 3-10), and the higher bands is the adapters loaded by the fusion protein (figure 10, lane 7-9). The presence of this upper band on figure 10, lane 9 indicates that applying heat to the fusion protein is not enough to destroy the complex. Lastly, the absence of upper band on the negative control where either the adapter (figure 10, lane 2) or the Tn5 (figure 10, lane 3,4 and 5) was omitted confirmed that the observed mobility shift on the other lanes are due to adapter loading on the Tn5.
To demonstrate that the observed upper bands are indeed the adapter within the fusion protein, the same gel were scanned with typhoon imager to visualize the Cy5 labelled adapter. The result is shown on figure 11. The presence of these upper bands under the typhoon imager (figure 11, lane 7-8) proved that the observed upper bands are indeed the adapters.
Part B: Target DNA mobility shift
A mobility shift in the target DNA is shown on figure 12. A mobility shift in the target DNA is observed for 804-1340nM fusion loaded with sgRNA (figure 12, lane 6, 14, and 15), however mobility shifts are not observed when the fusion is incubated with target DNA, but without sgRNA (figure 12, lane 8-11).These results indicating that the sgRNA loaded fusion can forms a complex with target DNA which has a complementary sequence to the sgRNA. Note the mobility shift occurs because the complex retards the movement of the DNA through the polyacrylamide gel, thus two distinct bands can be observed. The lower band at 632bp is the target DNA alone (figure 12, lane 2-15), and the higher band at >1000bp is the target DNA in the fusion complex (figure 12, lane 6, 14, and 15).
To demonstrate that the observed upper bands are indeed the target DNA in the fusion complex, the same gel was stained with silver stain to track the location of the protein instead of the DNA. The protein can be seen in lane (figure 13, 4-6, and 9-15), at the same location where the mobility shift of the target DNA was observed. This indicates that the DNA was indeed within the protein, further confirming the mobility shift. Additionally for the negative controls (figure 13, lane 9-11), protein is observed on the protein gel Figure 13 but DNA is not observed on the DNA gel figure 12, proving that without gRNA, the fusion cannot form a complex with the target DNA.
In vitro functionality testing: Integration assay
Aim
To prove the ability of the fusion protein to introduce targeted integration of the adapter next to the target sequence of sgRNA-dxCas9. When integration takes place, a double stranded break in the target DNA occurs, producing two fragments. The two fragments can be PCR amplified using a suitable primer sets that will bind to the beginning/end of the target sequence and to the end of the adapters. Each fragment is a unique sizes that can be visualized on a 5% TBE native PAGE.
Procedure
The in vitro functionality was tested according to the [http://2018.igem.org/Team:TUDelft/Experiments#tn5fusionintegrationassay integration assay protocol] on our wiki. To summarize, 2.5µM adapter was incubated with ~0 - 2.5µM fusion protein in reaction buffer (20 mM Tris-HCl, 100 mM KCl, 5% v/v glycerol, 1 mM beta-mercaptoethanol, 0.1mM EDTA, pH 7.5) for 1 hour at 23°C to load the adapter into the Tn5 portion of the fusion protein. Next, this complex was incubated with 0.6µM sgRNA for 10 minutes at 37°C to load the sgRNA into the dxCas9 portion of the fusion protein. Lastly, 10nM of target DNA was added and incubate for 1 hour at 37°C in the presence of 10mM MgCl2 to allow integration to occur [6];[8];[3]
Figure 14 depicted the experimental design for this functionality assay. Tn5 forms a dimer to pick up adapter DNA (50bp). This makes our fusion protein to consist of two Tn5 molecules and two dxCas9 molecules. We hypothesized that the loaded dxCas9 will steer the complex to a specific sequence of the synthetic EPO gene where the transposase will integrate the adapters. The resulting DNA fragments will be in specific sizes, which can be amplified by means of PCR and subsequently visualised on TBE native PAGE.
Table 5. The sequence list of the adapter, sgRNA (BBa_K2643013) and the target EPO (BBa_K2643004). Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.
To visualize the products of the integration, PCR was done using the following primer sets.
Table 6. Primer lists used for the functionality assay.
Figure 15 depicts the binding location of the primer sets on the expected products of the integration assay.
Each PCR reaction are loaded on separate lanes on 5% TBE native PAGE to visualise the amplification products shown on Figure 15.
Results
The functionality test was performed and the amplification product of PCR with primer sets Fw EPO + Rv adapter and Rv EPO + Rv adapter was visualized on 5% TBE native PAGE gel (figure 15). Thick bands around ~250bp and ~450bp (figure 15, lane 2 and 3) respectively indicate the amplification of the expected fragments after the integration (figure 14), this suggested targeted integration of adapter by our fusion protein. Additionally, different negative controls were done. The description of each negative control (figure 16, lane 4 to 13) are shown on figure 16. The absence of the ~250bp and ~450bp bands on the negative controls confirms that these bands are the result of the specific integration by our fusion protein.
The same experiment was done with longer incubation time. Figure 17 shows the amplification product of PCR with the same primer sets after 8 hours of integration. The position of the bands are comparable to the 1 hour incubation result (thick bands on ~250bp and ~450bp mark on figure 17, lane 2 and 3 respectively). However, it can be seen that the intensity of these bands decreases, while the intensity of other unspecific bands on the lane increases slightly (position 50bp - 150bp). This result suggested that unspecific integration event increases with longer incubation time. The same experiment were done with 4 and 16 hours incubation time. The results were consistent (increasing intensity of unspecific bands).
Summary
The fusion protein biobrick was fully characterized, from strain construction to in vitro functionality testing. The in vitro EMSA proved that the Tn5 portion of the fusion was able to load adapter. Moreover, it also showed that the dxCas9 portion of the fusion protein was able to load sgRNA and formed complex with the target DNA. The functionality of the fusion protein was tested by adapter integration assays. Using specific primers, PCR confirmed the target specific integration.
Source
Plasmid #60240 from Addgene, pTXB1-Tn5 from Picelli et al. [3], was used as template for Tn5 amplification. Plasmid #108383 from Addgene containing dxCas9 (3.7) from Dr. David Liu [1] was used as template for dxCas9 amplification.
Safety
This Biobrick contains the Tn5 transposase originating from Escherichia coli. Normally, a transposase is flanked by recognition sites called Mosaic Ends (MEs). This combination is called a transposon, which is a form of a mobile element. In this case, the Tn5 transposase is capable of recognizing these MEs, isolating the whole transposon and pasting it elsewhere. We made sure the coding sequence for the Tn5 transposase was not flanked by MEs at any time of the project. This was done to prevent uncoordinated migration of the transposon. For a similar reason, we verified the absence of genomic MEs with the genome of the host strains, to prevent uncontrollable scrambling of a host’s genome. The only sequences that contained MEs in this project were linear fragments that were supplied to a host through transformation. Through this setup, we controlled as much as possible to make working with Tn5 transposase as safe as possible and sufficient to work under ML-1/BSL-1 laboratories.
Additionally, 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.
References
- ↑ 1.0 1.1 1.2 1.3 Hu J.H., Miller S. M., Geurts M. H., Tang W., Chen L., Sun N., Zeina C. M., Gao X., Rees H. A., Lin Z., Liu D. R. (2018). Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature; 556(7699): 57–63. doi: 10.1038/nature26155
- ↑ Adey, A., Morrison, H. G., Asan, Xun, X., Kitzman, J. O., Turner, E. H., … Shendure, J. (2010). Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biology, 11(12), R119. http://doi.org/10.1186/gb-2010-11-12-r119
- ↑ 3.0 3.1 3.2 3.3 3.4 3.5 Picelli, S., Björklund, Å. K., Reinius, B., Sagasser, S., Winberg, G., & Sandberg, R. (2014). Tn5 transposase and tagmentation procedures for massively-scaled sequencing projects. Genome research, gr-177881.
- ↑ 4.0 4.1 Rueda, F.O., Bista, M., Newton, M.D., Goeppert, A.U., Cuomo, M.E., Gordon, E., Kroener, F., Read, J.A., Wrigley, J.D., Rueda, D.L., & Taylor, B.J. (2017) Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease. Nature Communications. 8(1), 1610. DOI: 10.1038/s41467-017-01732-9
- ↑ 5.0 5.1 Huai, C., Li, G., Yao, R., Zhang, Y., Cao, M., Kong, L., Jia, C., Yuan, H.J., Chen, H., Lu, D., & Huang, Q. (2017) Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Nature Communications, 8(1), 1375. DOI:10.1038/s41467-017-01496-2.
- ↑ 6.0 6.1 6.2 6.3 Sternberg, S.H., LaFrance, B., Kaplan, M., Doudna J.A. (2015) Conformational control of DNA target cleavage by CRISPR–Cas9. Nature; 527(7576): 110–113. Published online 2015 Oct 28. doi: 10.1038/nature15544
- ↑ 7.0 7.1 Whitfield, C. R., Wardle, S. J., & Haniford, D. B. (2008). The global bacterial regulator H-NS promotes transpososome formation and transposition in the Tn5 system. Nucleic acids research, 37(2), 309-321.
- ↑ Hennig, B. P., Velten, L., Racke, I., Tu, C. S., Thoms, M., Rybin, V., . . . Steinmetz, L. M. (2017). Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3, 8(1), 79-89. doi:10.1534/g3.117.300257
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