Part:BBa_K4308008
CasΦ_crRNA
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 some crRNA in our CasΦ system.
Biology
The insertion or deletion of uridine residues into kinetoplastid during RNA editing is a small non coding RNA that can pair with pre mRNA. crRNA edits RNA molecules, about 60-80 nucleotides in length, and is transcribed by a single gene.
Usage
By combining with CasΦ, crRNA participates in the target recognition and cleavage of CasΦ system.
Characterization
1.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[2]. 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. 1 The sequence of crRNAs with hairpin structures.
Figure. 1 The reaction rates of FQ reporter cleavage by Mut-4 with hairpin structure crRNA.
2. The ssDNA cleavage activity of CasΦ-crRNA
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. 2 PAGE results for cleavage ssDNA target with different Cas-crRNA.
3. The trans-cleavage activity of CasΦ-crRNA
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 2.
Table. 2 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-crRNA might regulate the accessibility of the RuvC domain for the association of single-stranded DNA (ssDNA).
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 different mutants.
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 wild-type CasΦ-crRNA 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-crRNA can be almost ignored, indicating that CasΦ-crRNA 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-crRNA nucleases 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. 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. 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).
[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]
- 1000COMPATIBLE WITH RFC[1000]
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