Part:BBa_K4308036
CasΦ(Neg-K)-crRNA-LINC00857
CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing[1]. For our target LINC00857, we designed crRNA in our CasΦ system.
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
Based on the trans-cleavage ssDNA activity, Doudna et al. have proved that CasΦ can be applied for fluorophore quencher (FQ) reporter assay for nucleic acid detection [1]. Whether for in vivo genome editing or in vitro nucleic acid detection, the ability of Cas nuclease to recognize mismatch targets is very important. Compared with Cas12a, CasΦ shows better mismatch target recognition capability. When a single-base mismatch is located in the centre of the target recognition region, CasΦ nuclease shows a good substrate specificity for matched and mismatched targets.
By combining with CasΦ, H4 participates in the target recognition and cleavage of CasΦ system. Compared with normal crRNA, the specificity of CasΦ-H4 system increased.
Characterization
1. Proof of the expression
We used SDS-PAGE to verify the existence of CasΦ and CasΦ(Neg-K). During the purification procedure, we used nickel column to purify the protein. After the column was balanced, add 30mM, 60mM, 150mM 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 protein were detected in 30mM, 60mM and 300mM imidazole buffer.Shown in Figure 2, the results indicated that Neg-K protein were detected in 30mM, 60mM and 300mM imidazole buffer.
Figure. 1 SDS-PAGE results for expression of CasΦ.Lane 1: CasΦ in imidazole buffer (300 mM); Lane 2: CasΦ in imidazole buffer (300 mM); Lane 3: CasΦ in imidazole buffer (60 mM); Lane 4: CasΦ in imidazole buffer (30 mM).
Figure. 2 SDS-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
We verified the cleavage activity of the CasΦ with the ssDNA target, which can only be partially changed. The PAGE results are as shown in Figure 2.
Figure. 3 PAGE results for cleavage ssDNA target with different Cas-crRNA.
3. The trans-cleavage activity of CasΦ
The fluorophore quencher (FQ) reporter assays were employed to evaluate the target-triggered trans-cleavage activity of wild-type CasΦ. 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, which further verify that the helix α7 of CasΦ might regulate the accessibility of the RuvC domain for the association of single-stranded DNA (ssDNA).
Figure. 4 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.
Figure. 5 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 wild-type CasΦ 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 wild-type CasΦ can be almost ignored, indicating that CasΦ 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), CasΦ nucleases produce non-specific signals that were comparable with the complementary target.
Figure. 6 The reaction rates of FQ reporter cleavage by Cas-crRNA in the presence of DNA targets with single-base mismatch.
5. Effect of crRNA secondary structure on specificity of CRISPR-Cas system
As reported by Gersbach et al. a hairpin secondary structure onto the spacer region of crRNA can increase the CRISPR-Cas system specificity for target cleavage. The FQ-reporter assays showed that the recognition of complementary target or mismatch target by CasΦ-crRNA was affected by the hairpin structure in crRNA at the same time.
Table. 2 The sequence of crRNAs with hairpin structures
Figure. 7 The reaction rates of FQ reporter cleavage by Mut-4 with hairpin structure crRNA.
6. 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. For WT/CrRNA system, only when the fraction of target sequences was more than 10% can the signal differentiated from the mismatch sequences be generated.
Figure. 8 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).
[2] Kocak DD, Josephs EA, Bhandarkar V, Adkar SS, K. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat Biotechnol 37, 657-666(2019).
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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