Part:BBa_K4308016
CasΦ(v)
CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing[1].CasΦ(v) is a faster variant of CasΦ(v) designed by Doudna’s group.[2]
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 Δ155–176 (GSSG). It is designed by Doudna’s group and was proved to be a faster variant[1]. Also, the mismatch tolerance profiles of CasΦ(v) are comparable to wild-type CasΦ.
Usage
CasΦ(v) 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(v).
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 CasΦ(v) protein were detected in 30mM, 60mM and 300mM imidazole buffer.
Figure. 1SDS-PAGE results for expression of CasΦ(v).Lane 1: CasΦ(v) in imidazole buffer (300 mM); Lane 2: CasΦ (v) in imidazole buffer (300 mM); Lane 3: CasΦ(v) in imidazole buffer (60 mM); Lane 4: CasΦ(v) in imidazole buffer (30 mM).
2. The trans-cleavage activity of CasΦ(v)
The fluorophore quencher (FQ) reporter assays were employed to evaluate the target-triggered trans-cleavage activity of CasΦ(v). 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 2. The activity of CasΦ(v) which was designed by Doudna’s group, expressed and purified by our team, and its results were consistent with its reported faster cleavage activity.
Figure. 2 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 different mutants. As shown in Figure 4, at all target concentrations, the trans-cleavage activities of CasΦ(n) were significantly higher than that of the wild-type CasΦ. At the medium concentrations (2-25 nM), the reaction rates of CasΦ(n) were higher than that of Neg-K.
Figure. 3 The reaction rates of FQ reporter cleavage by Cas-crRNA in the presence of DNA targets with different concentrations.
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).
[2] Pausch, P., K.M. Soczek, D.A. Herbst, et al. DNA interference states of the hypercompact CRISPR-CasPhi effector. Nat Struct Mol Biol 28, 652-661, doi: 10.1038/s41594-021-00632-3(2021).
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 971
Illegal BsaI.rc site found at 1363
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