Part:BBa_K4308007
Cas12a1_crRNA
Biology
As the description of iGEM18_TJU_China, Cas12a (BBa_K2644102) can be applied for fluorophore quencher (FQ) reporter assay for nucleic acid detection based on the trans-cleavage ssDNA activity. In vitro nucleic acid detection, it is often necessary to consider the specific cleavage rate of Cas nuclease. Cas12a has high cleavage efficiency for both target double-stranded DNA and non-target single-stranded DNA, but its ability to recognize SNP is not as good as CasΦ. Cas12a was used as a more mature control in this project to assist us in selecting appropriate Cas nucleases.
Characterization
1. The dsDNA cleavage activity of Cas12a
We verified the cleavage activity of the Cas12a with the dsDNA target, which can only be partially changed. We mixed Cas12a targeting dsDNA and Cas12a and kept them warm at 37 °C. We performed three sets of biological replicates. The PAGE results are showed in Figure 1.
Figure. 1 PAGE results for cleavage dsDNA target with Cas12a-sgRNA.The red box in lane 1 shows the band cut by Cas12a-sgRNA.
Figure. 2 The ssDNA cleavage activity of Cas14a. Line 1: Cas 14a+sgRNA+dsDNA; Line 2: Cas14a+dsDNA; Line 3: sgRNA+dsDNA; Line 4: ssDNA; Line 5: Cas 12a+sgRNA+dsDNA; Line 6: Cas12a+dsDNA; Line 7: sgRNA+dsDNA; Line 8: ssDNA.
2. The trans-cleavage activity of Cas12a
The fluorophore quencher (FQ) reporter assays were employed to evaluate the target-triggered trans-cleavage activity of Cas12a. The final reaction (20 μl) contained final concentrations of 100 nM Cas12a, 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 37C. The sequence of FQ probe were listed in Table 1.
Table. 1 The sequence of 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 Cas12a has high cleavage efficiency for non-target single-stranded DNA.
Figure. 3 Results of the FQ-reporter assays.
3. Active contrast
We compared the activities of Cas12a, Cas14a, CasΦ, CasΦ (Neg-K), using fluorophore quencher (FQ) reporter assays (Figure 5). Cas12a can cleave dsDNA faster and exert non-specific cleavage activity against ssDNA. Despite its high activity, Cas12a has poor SNP recognition ability.
Figure. 4 The reaction rates of FQ reporter cleavage of Cas12a, Cas14a,CasΦ and CasΦ Neg-K systems.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 18
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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