Part:BBa_K4308017
CasΦ(Neg-K)
CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing[1].CasΦ(Neg-K) is a faster variant of CasΦ designed by our group.
Biology
The protein size of CasΦ is 70 to 80 kDa, about half the size of Cas9 and Cas12a, but maintains the ability to unwind and cut dsDNA. Cryo-EM-based structural studies indicate that CasΦ forms a compact structure, in which protein and crRNA are interwoven to realize RNA-guided dsDNA unwinding and cleavage. CasΦ also exhibits target-activated trans-cleavage ssDNA activity, which is an activity associated Cas12 nucleases family. This CasΦ mutant went through mutations of E159K, S160K, S164K, D167K, E168K. It is designed by our group and was proved to be a more effective variant. Also, the mismatch tolerance profiles of Neg-K are comparable to wild-type CasΦ.
Usage
Neg-K can be used for more efficient nucleic acid detection in vitro and could be similarly engineered for more sensitive and ‘time-saving’ in vitro diagnostics.
Characterization
1. Proof of the expression
We used SDS-PAGE to verify the existence of CasΦ(Neg-K).
During the purification procedure, we used nickel column to purify the protein. After the column was balanced, add 30mM, 60mM and 300mM imidazole buffer respectively for flushing. We collected when the chromatographic column curve showed an upward trend, and stop collecting when the curve tended to be flat. Each concentration of imidazole buffer solution was collected for sample preparation, run SDS-PAGE electrophoresis, dye and decolor. The concentration represented by the sample with the target band is concentrated by ultrafiltration.Shown in Figure 1, the results indicated that Neg-K protein were detected in 30mM, 60mM and 300mM imidazole buffer.
Figure. 1SDS-PAGE results for expression of CasΦ(Neg-K).Lane 1: CasΦ(Neg-K) in imidazole buffer (300 mM); Lane 2: CasΦ(Neg-K) in imidazole buffer (60 mM); Lane 3: CasΦ(Neg-K) in imidazole buffer (30 mM).
2. The ssDNA cleavage activity of CasΦ(Neg-K)
We verified the cleavage activity of Neg-K with the ssDNA target. Single-strand DNA labelled with fluorescence at the end was incubated with different mutant proteins. The PAGE results are as shown in Figure 2. Under the catalysis of Neg-K, the target DNA was apparently cleavaged. From the preliminary results of PAGE, there was no significant difference between Neg-K and the wild-type protein in the cleavage activity of the single-strand DNA target.
Figure. 2 PAGE results for cleavage ssDNA target with different Cas-crRNA.
3. The trans-cleavage activity of CasΦ(Neg-K)
The fluorophore quencher (FQ) reporter assays were employed to evaluate the target-triggered trans-cleavage activity of wild-type CasΦ(n). The final reaction (20 μL) contained final concentrations of 100 nM CasΦ, 120nM crRNA, 100nM FQ probe, with 50 nM target DNA in cleavage buffer (10 mM HEPES-Na pH7.5, 150 mM KCl, 5 mM MgCl2, 10% glycerol, 0.5 mM TCEP). Fluorescence signals were obtained every 2 minutes at 37°C. The sequence of crRNA, activator ssDNA and FQ probe were listed in Table 1.
Table. 1 The sequence of crRNA, target DNA and FQ probe for FQ-reporter assays.
The results of the fluorescence analysis were shown in Figure 3. Neg-K has a high target triggered trans-cleavage activity compared to the wild-type. And it has comparable cleavage activity to the CasΦ(n) designed by Doudna's group.
Figure. 3 The time-course fluorescence intensity curves of FQ reporter cleavage by different Cas-crRNA in the presence of DNA targets.
Further, the DNA detection performances of mutants were investigated by a series of DNA targets with different concentrations. The initial reaction rate of the fluorescence signal was employed to evaluate the trans-cleavage activity of Neg-K. As shown in Figure 4, at all target concentrations, the trans-cleavage activities of Neg-K were significantly higher than that of the wild-type CasΦ. But there was no significant difference between nCasΦ and Neg-K. At high (50 nM) or low (1.25 nM) concentration, the initial reaction rate of Neg-K was higher than that of nCasΦ. Although this conclusion still needs more experimental data to be proved, the preliminary results show that the Neg-K constructed by our team has a good trans-cleavage activity for DNA detection.
Figure. 4 The reaction rates of FQ reporter cleavage by Cas-crRNA in the presence of DNA targets with different concentrations.
4. Specificity for single-base difference
In order to test the recognition ability of Neg-K to single-base difference targets, we introduced a single-base mismatch at different positions in the target sequences (Table 1). As shown in Figure 5, when the single-base mismatch was at position 11 or 12 (number from 3 'end), the nonspecific signals produced by Neg-K can be almost ignored, indicating that Neg-K has high recognition specificity for single-base mismatch at these positions. This may be due to the reduced stability of the crRNA/DNA hybrid when the single-base mismatch is located in the middle region of the crRNA and DNA target hybridization. However, when the single-base mismatch was at position 13 (number from 3 'end), Neg-K produce non-specific signals that were comparable with the complementary target.
Figure. 5 The reaction rates of FQ reporter cleavage by Cas-crRNA in the presence of DNA targets with single-base mismatch.
5.Performance of optimized system for DNA mutation detection
In order to verify the performance of our optimization CRISPR-Cas system for picking up DNA mutations from a large number of background sequences, we mixed different amounts of target sequences with mismatch sequences (MT13) to simulate artificial samples containing 50% to 0% DNA mutations. We chose Neg-K (a CasΦ mutant designed by our group) and crRNA H4 to construct the mutation detection system. As shown in Figure 6, compared with the conventional wild-type CasΦ - hairpin free crRNA system (WT/CrRNA in Figure 6), the Mut-4/H4 system showed better mutation detection performance.For Mut-4/H4, even if the fraction of target sequences was as low as 2%, the initial reaction rate of the fluorescence signal was still higher than that of the mismatch sequences.
Figure. 6 The reaction rates of FQ reporter cleavage with samples containing 50% to 0% DNA mutations.
References
[1] Pausch, P., B. Al-Shayeb, E. Bisom-Rapp, et al. CRISPR-CasΦ from huge phages is a hypercompact genome editor. Science 369, 333-337, doi: 10.1126/science.abb1400(2020).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1022
Illegal BsaI.rc site found at 1414
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