Part:BBa_K4223008

Cas14a1 protein expression system

CRISPR-Cas14 protein, which is found almost exclusively in the superphylum of Archaeophilus. CRISPR-Cas14 protein has the activity of targeting single stranded ssDNA, and it is twice smaller than CRISPR-Cas9 protein.
Combining the nonspecific ssDNase cleavage activity of CRISPR Cas14 protein with an isothermal amplification method (DETECTR-Cas14), it is also promising to be developed for high-fidelity DNA single nucleotide polymorphism genotyping to detect ssDNA viruses, with potential clinical, ecological, and economic importance. Thus, CRISPR-Cas14 could play a huge role in CRISPR diagnostics for both infectious and noncommunicable diseases.
Based on previous research, we developed a single-base, amplification-free, portable nucleic acid detection platform based on Cas14a1 with high fidelity, high specificity, and high efficiency.

1.Solution required for the experiment

(1) LB (Luria-Bertani) liquid medium.
1% (w/v) Tryptone, 1% (w/v) NaCl, 0.5% (w/v) Yeast Paste.
(2) TEV and Cas14a1 affinity chromatography (Ni column) purification solutions.
Lysis solution: 10 mM imidazole, 500 mM Nacl, 20 mM Tris-Hcl, 5% glycerol, 5 mM β-mercaptoethanol.
Equilibration solution: 0 mM imidazole, 500 mM Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.
Elution solution: 300 mM imidazole, 500 mM Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.
(3) Cas14a1 heparin column purification solution.
Equilibrium solution: 100 mM Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.
Elution solution: 2 M Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.

2.Plasmid Transformation and Protein Induced Expression

Transformation of MBP-Cas14a1 plasmid into DE3 cells was performed as follows: DE3 receptor cells frozen at -80°C were removed and thawed naturally in an ice box (DE3 cells should be used once to avoid reuse after freezing), 1 μL of the above plasmid was aspirated and mixed homogenously with 50 μL of DE3, placed on ice for 30 min, transferred to 42°C for 90 sec and then immediately placed on ice for 3 min. Immediately place on ice for 3 min. add 700 μL LB medium (without antibiotics) and resuscitate for 1 hr at 37°C 180 rpm (all cultures were at this shaker speed and will not be repeated) under aseptic conditions. The successfully transformed E. coli was centrifuged at high speed, 700 μL supernatant was discarded and the remaining bacterial solution was mixed, and 30 μL was evenly coated with LB solid medium containing 50 μg/mL ampicillin (AMP) using a spreading rod and incubated in a light incubator at 37°C for 12 h. The monoclonal bacteria were then picked and cultured in 10 mL EP tubes, and the monoclonal strain was incubated overnight in 20% glycerol. After overnight culture, the monoclonal strains were stored in 20% glycerol for freezing.

Through literature review, we found that the induced expression of Cas14a1 increased and then decreased with the increase of isopropyl-β-D-thiogalactoside (IPTG) concentration, and the expression was higher at 0.2 mM IPTG, and the best induction was achieved at the temperature of 18°C.

Figure 1. SDS-PAGE to detect the expression of Cas14a1 under different inducer concentration (a) and different induction temperature (b)

Therefore, we added glycerobacteria containing Cas14a1 plasmid at a final concentration of 1% to 10 mL EP tubes containing AMP and LB medium for a small amount of propagation culture, and 12 h later transferred fresh bacterial solution from EP tubes at the same 1% in 1000 mL conical flasks at 37°C, 180 rpm, and carried out a large amount of propagation culture until OD600 was 0.6. 10His-MBP- Cas14a1 protein was expressed at 18°C, 180 rpm, and induced overnight by adding 0.2 mM IPTG to a final concentration of 0.2 M.

3.Cas14a1 protein purification by affinity chromatography (Ni column)

Cells were centrifuged at 10,000 rpm for 10 min at 4°C. The pellet was collected, resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM DTT, 0.2 mM PMSF, 5% glycerol, 5 mM β-mercaptoethanol, 10 mM imidazole), and broken up by ultrasonication. The temperature was lowered to 4°C, and cell pellets were removed by centrifugation at 8000 rpm and 30 min. The protein supernatant was added to Bio-Scale Mini Nuvia IMAC Ni - charge (Bio-Rad laboratories, Inc., USA) in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM β-mercaptoethanol buffer with increasing concentrations of imidazole (from 10 to 300 mM). eluted with a linear gradient (refer to the following content (7)).

Cas14a1 affinity chromatography (Ni column) purification: The Cas14a1 purification procedure is divided into 7 steps: cleaning, equilibration, sample loading, washing, elution, water washing and 20% ethanol filling of the column. (1) Washing the Ni column with pure water (A1 channel) at a flow rate of 7 mL/min for 10 volumes. (2) Equilibrating the Ni column with 98% equilibration solution (A2 channel) + 2% eluent (B channel) at a flow rate of 7 mL/min for 5 volumes. (3) Protein loading (spiked channel) at a flow rate of 1 mL/min for 120 mL. (4) Washing the Ni column with equilibration solution (A2 channel) for 5 volumes. (5) Gradient elution of target protein in eluent (B channel), flow rate 3 mL/min, 6 volume for 0-30% elution; 10 volume for 30-40% elution; 5 volume for 40%-60% elution; 5 volume for 60%-70% elution; 6 volume for 70%-100% elution; 6 volume for 100%-100% elution. 6 volume; 100%-100% elution 10 volume. (6) Pure water (A1 channel) washed the residual solution in the purification column with a flow rate of 7 mL/min and 6 volume. (7) 20% ethanol (A3 channel) cleaned the purification column with a flow rate of 5 mL/min and eluted 5 volume, filled with ethanol, unloaded the column and immersed in 20% ethanol, and stored at 4 ℃ for proper storage. The role of low temperature and 20% ethanol is to avoid the growth of bacteria, which affect the re-use.

The target protein was purified by using the protein His-tag to chelate with nickel in the affinity chromatography column to enrich the target protein, and then the protein was eluted by imidazole competition. The results of SDS-PAGE characterization of Cas14a1 are shown in Figure 2. The appearance of a single band near 108 kDa, consistent with the theoretical molecular weight of Cas14a1 (Figure 2a), indicates that the protein was successfully purified and ready for subsequent experiments.

Figure 2. SDS-PAGE detection Cas14a1 (a) Ni column purification
M: Protein Marker; 1-4: indicate the number of tubes for collecting Cas14a1 protein fractions.

4.TEV protease cleaved MBP tag

TEV enzyme was homogeneously mixed with Cas14a1 protein at a 1:1 concentration ratio and then digested at 4°C for 12 h. The 10×His- MBP label was removed for subsequent heparin column purification.

The digestion results were verified by SDS-PAGE gel electrophoresis assay. As shown in Figure 3, TEV was added to the fusion protein Mbp-Cas14a1 (108 kDa), and the theoretical size band at 108 kDa disappeared after 12 h, while the excised labeled Cas14a1 band appeared at 63 kDa, indicating that the enzymatic cleavage was relatively complete. The mixed proteins after digestion were continued to be purified by heparin column.

Figure 3. SDS-PAGE detection of TEV digestion MBP-Cas14a1: uncut Cas14a1 protein; +TEV: 12h after digestion; Marker: protein rainbow Marker.

5.Heparin column purification of Cas14a1 protein

For this part of the experiment we took advantage of the affinity of Cas14a1 protein for heparin. After concentrating the Cas14a1 protein solution, we replaced the imidazole in the protein solution with a heparin column equilibrator to remove it, and then we cleaned the ultrafiltration tubes and other related equipment. The protein concentrate was filtered through a 0.22 µm filter and the protein was purified again on a GE HiTrap™ Heparin HP (HUIYAN BIO) column, and the protein fraction containing Cas14a1 was collected at the λ (280 nm) absorption peak.

The detailed steps and procedures for heparin column purification were as follows: ① High flow rates can cause column pressure to exceed the limit, so a flow rate of 1 mL/min was used throughout the heparin column after installation. First, manually clean the A, B and loading paths of the machine with pure water at a flow rate of less than 10 mL/min. After each channel λ (280 nm) is balanced, install the heparin column and operate carefully to avoid bubble generation. The heparin column was washed with pure water (A1 channel) in a volume of 10 volume. ③ The heparin column was equilibrated with 98% equilibration solution (A2 channel) + 2% eluent (B channel) in a volume of 16 volume. ④ Protein loading (loading channel) in a volume of 50 mL. ⑤ Equilibration solution (A2 channel) washed with miscellaneous proteins in a volume of 16 volume. ⑥ The target proteins were eluted with a gradient of eluent (B channel) in 0-10%, 10-40%, 40-55%, 55-60%, 60-80%, 80-100%, and 100-100% in elution volumes of 5, 5, 8, 5, 4, 6, and 15 volume. eluted in a linear gradient with increasing NaCl concentration (from 100 mM to 2M).

After completing the above steps, we used SDS-PAGE gels to detect Cas14a1 protein fractions and also used a gel imaging system to view the results.

Figure 4. SDS-PAGE detection heparin column purification
M: Protein Marker; 1-5: indicates the number of tubes for collecting the target protein fraction.

6.Protein cleavage activity validation

To verify Cas14a1 cleavage activity, one experimental group (Cas14a1+sgRNA+ssDNA target) and three control groups were set up. The fluorescence assay was performed at 37℃ using an enzymatic standard (excitation wavelength 492 nm, emission wavelength 520 nm) for the 4 groups of samples. The variance and mean of 3 parallel samples for each group of samples were calculated and plotted with Origin 9.0 as shown in Figure 5 Only the experimental group had elevated fluorescence and it was very obvious (fluorescence value close to 105). No fluorescence signal was generated when any of Cas14a1, guide RNA (sgRNA), or target ssDNA was missing from the assay system, excluding the fluorescence elevation due to other reasons and demonstrating the high cleavage activity of Cas14a1 protein.

Figure 5. Cas14a1 cleavage activity verification

7.Confirmation of “false positive” characterization

Cas14a protein is similar to Cas13a protein in that it tolerates 1-2 mismatches between crRNA and target sequence, which also leads to a significant reduction in the cleavage efficiency of Cas14a protein and results in a "false positive" characterization in the assay.

To confirm the occurrence of false positives, HainanU-China designed a series of sequences with single nucleotide polymorphisms (SNPs) based on the target RNA and used them for fluorescence reporter detection.

It should be noted that the fluorescence is generated by the trans cleavage of the ssDNA-FQ probe after Cas14a recognizes and cleaves the target DNA.

To provide data support for the false positive characterization, we introduced 13 DNAs, including 1 Target and 12 DNA sequences containing single base mutations at odd sites. The specific mutation sites are shown in Figure 6.

Figure 6. Single nucleotide polymorphisms(SNPS) at odd loci in 12 different DNA and Target DNA

The recognition and cleavage by Cas14a1 protein of the artificially designed RNA, thus exhibiting the trans cleavage activity and consequently the fluorescence signal report, we show as Relative fluorescence unit (RFU). We can observe that each detected sequence exhibits a different fluorescence signal intensity, and RM8, RM15 and RM19 are significantly larger than the target sequences. This indicates that false positives do exist and have different effects on the specificity of the assay depending on the mutation site.

Figure 7. Detection results of Cas14a protein for DNA sequences (revealed by relative fluorescence unit RFU)

8.Verification of clamp effect

Clamp is a system we envisioned to assist Cas13a and Cas14a proteins to avoid "false positive" characterization caused by single base mutation sequences by artificially designing complementary PNAs (peptide nucleic acids) of single base mutation sequences to shield them from interference in the assay system.
Taking Cas14a1 as an example, based on the experimental results of Step 2 (Confirmation of "false positive" characterization), sequences with mutation sites at 5'-5, 7, 20-3' bases were selected from a series of sequences with single-base mutations. 5'-5, 7, 20-3' bases, which showed high RFU in the false positive mock assay, were selected as typical sequences to demonstrate the role of the shackle system (clamp).
Initially we used the designed complementary DNA as clamp as a pre-experiment to verify the preliminary role of Clamp. As illustrated by the experimental results presented in Fig 8: the Relative fluorescence unit (RFU) of the experimental group with the addition of DNA-clamp was significantly lower than that of the control group, and combined with the Delta-RFU analysis, Clamp did reduce the interference of single-base mutant sequences on the assay results.

Figure 8. The Relative fluorescence unit (RFU) of the experimental group with the addition of DNA-clamp

However, DNA as clamp alone is not enough for the goal of our project. So we discovered peptide nucleic acids (PNA), a class of DNA analogs with a peptide backbone replacing the sugar phosphate backbone, as nucleic acid sequence-specific reagents by reviewing the literature [2]. We designed PNA as clamp and validated it in the same way (Figure 9). We also compared the control group with DNA-clamp and PNA-clamp to make the data more reliable (Figure 10).
The above two experiments also demonstrated that our idea of designing "Clamp" to avoid the shortcoming of Cas13a and Cas14a in detecting the target sequences due to similar mutated sequences, which leads to misidentification, can be realized.

Figure 9. The Relative fluorescence unit (RFU) of the experimental group with the addition of PNA-clamp

Figure 10. Comparison of DNA-clamp and PNA-clamp

Although we have verified that PNA as clamp has better effect than DNA as clamp, however, we are not yet sure to what extent the shielding effect of using PNA as clamp on single base mutation false sequences can be achieved, and it is not clear what effect different concentrations of PNA have on the effect of target sequence detection. So, we set a certain amount of PNA (100nM) and gradient concentrations of target and mutant sequences in combination, and found that the interference of PNA on mutant sequences was significantly greater than that of target sequences, and even after the concentration of mutant sequences was less than 100nM, its RFU dropped in a precipitous manner. When PNA was relatively saturated in the mutant sequence, it was able to play an almost complete shielding role. In the absence of PNA addition, the magnitude of RFU change of both was not as obvious as when PNA was added (Figure 11).

Figure 11. Concentration gradient series experiments

Through further experimental design and verification, we found that the shielding effect of PNA on mutant sequences at a certain concentration of PNA (100 nM) was more obvious at lower concentrations (<100 nM), and the shielding effect of PNA gradually diminished as the concentration of mutant sequences increased (Figure 12). It can be seen that the interference rate of PNA on mutant sequences is gradually reduced with the increase of the latter concentration until it tends to 0 (Figure 12).

Figure 11. Experiments with certain concentrations of PNA-CLAMP

Sequence and Features

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