Part:BBa_K4308009
Cas12f_crRNA
Cas14a，also known as Cas12f, is a kind of endonuclease that specifically binds to and cleaves the target dsDNA under the guidance of sgRNA (PAM site is required). As the description of iGEM21_IISER_Berhampur, Cas14a (BBa_K3982001) can bind to the target nucleic acid and activate its ssDNA trans-cleavage activity, so it has been applied to the molecular detection of the target nucleic acid in our project.
Biology
Recent studies identified type V-F Cas14a (Figure 1) proteins as exceptionally compact RNA-guided DNA endonucleases. Cas14a is consist of 400~700 amino acid residues and much smaller than Cas9 and Cas12 (950~1400 amino acids). The Cas14a from an uncultured archaeon is consist of 529 residues and lacks detectable sequence identity with any other known proteins, except for the presence of the RuvC domain. Despite its small size, Cas14a can associate with an sgRNA and cleaves dsDNA targets with TTTR (where R is A or G) PAMs.
Figure. 1 Protein structure of Cas14a.[1]
Usage
Cas14a plays an important role in our diagnosis system. In our design, Cas14a was directed by sgRNA to cleave a partial fragment of LINC00857 amplified by RT-RPA. This process activates the activity of Cas14a to cleave non-target single-stranded DNA, which could cleave the DNA nanostructure we designed to release the γ-amylase embedded in it. Cas14a was involved in the cleavage of target double-stranded DNA and non-target single-stranded DNA, which is one of the core parts of our diagnostic system (Figure 2).
Figure. 2 Cas14a is involved in the core steps.
Characterization
1. Proof of the expression
Considering that Cas14a may form inclusion body in the engineered bacteria, so that the synthesized one would sink in the form of crystals, we added MBP tag to the N-terminus of Cas14a to make Cas14a soluble and convenient for purification.
We used SDS-PAGE to verify the existence of Cas14a. During the purification procedure, we used nickel column to purify the protein. After the column was balanced, we add 30mM, 60mM, 150mM and 500mM imidazole buffer respectively for flushing. We collected the washing water when the chromatographic column curve showed an upward trend, and stop it when the curve tended flat. Each concentration of imidazole buffer solution was collected for sample preparation, run SDS-PAGE electrophoresis, dyed and decolored. The concentration represented by the sample with the target band was concentrated by ultrafiltration. Show in Figure 3, the results indicated that CasΦ was detected in imidazole buffer of different concentrations. After purification, MBP tag was removed by protease cleavage to obtain biologically active Cas14a, which was verified by SDS-PAGE electrophoresis (Figure 4).
Figure. 3 SDS-PAGE results for expression of MBP-Cas14a.
Figure. 4 SDS-PAGE results for expression of Cas14a.
2. The ssDNA cleavage activity of Cas14a
We verified the cleavage activity of the Cas14a with the ssDNA target, which can only be partially changed. The PAGE results are shown in Figure 5.
Figure. 5 The ssDNA cleavage activity of Cas14a.Line 1: Cas 14a+sgRNA+dsDNA; Line 2: Cas14a+dsDNA; Line 3: sgRNA+dsDNA; Line 4: ssDNA.
3. ssDNA trans-cleavage activity
The fluorophore quencher (FQ) reporter assays were employed to evaluate the ssDNA trans-cleavage activity. The final reaction (20 μl) contained final concentrations of 100 nM Cas14a, 120nM sgRNA, 100nM FQ probe, with different concentration of 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. (Figure 6)
This verifies that it has sufficient sensitivity to identify targets at various concentrations, especially from 0.5nM to 25nM. Plus, the quantification ability of Cas14a excel within 30min. As a result, Cas14a is capable to complete the precise quantification of our system.
Figure. 6 The ssDNA trans-cleavage activity of FQ reporter cleavage by Cas14a in the presence of DNA targets with different concentrations.
Despite the significant improvement in cleavage rate and specificity of the Neg-K/H4 system compared to the WT/CrRNA system, the activity of the Neg-K /H4 system is still poor compared to the more mature Cas12a and Cas14a systems. This may be due to the small size of CasΦ, which is only half that of Cas12a, and therefore its enzymatic activity is also relatively low. Although Cas14 is also small in size, it remains highly active when it is cleaved because it is a dimer.
Figure. 7 The dsDNA trans-cleavage activity of FQ reporter cleavage by Cas proteins in the presence of DNA targets with different concentrations.
4. 4. Itegrating Cas14a into our diagnosis system
We reacted Cas14a and different concentration of dsDNA (copies of LINC00857)with the DNA nanostructure embedded with γ-amylase and amylose, and determined the final glucose concentration released after a period of time(5min/10min).The between-group difference was more pronounced for 5min reaction(Figure 8 and Figure 9 ).
Figure. 8 Reacting for 5min.
Figure. 9 Reacting for 10min.
References
[1] Takeda SN, Nakagawa R, Okazaki S, Hirano H, Kobayashi K, Kusakizako T, Nishizawa T, Yamashita K, Nishimasu H, Nureki O. Structure of the miniature type V-F CRISPR-Cas effector enzyme. Mol Cell 81, 558-570, doi: 10.1016/j.molcel.2020.11.035 (2020).
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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