Part:BBa_K4223004
Cas14a protein-coding gene
CRISPR-Cas14 protein, which is found almost exclusively in the superphylum of Archaeophilus. CRISPR-Cas14 protein has the activity of targeting single stranded ssDNA, and it is twice smaller than CRISPR-Cas9 protein.
Combining the nonspecific ssDNase cleavage activity of CRISPR Cas14 protein with an isothermal amplification method (DETECTR-Cas14), it is also promising to be developed for high-fidelity DNA single nucleotide polymorphism genotyping to detect ssDNA viruses, with potential clinical, ecological, and economic importance. Thus, CRISPR-Cas14 could play a huge role in CRISPR diagnostics for both infectious and noncommunicable diseases.
Description
Unique features of CRISPR-cas14a
The RNA mass of CRISPR Cas14a protein assembly complex was 48%, CRISPR Cas9 protein 17% and Cas12a protein 8%, respectively. RNA plays an important role in CRISPR Cas14a protein structure.
The CRISPR-Cas14a protein does not need to recognize PAM sites in the substrate DNA, while the CRISPR-Cas9 protein needs to recognize PAM rich in guanine and the CRISPR-Cas12a protein needs to recognize PAM rich in thymidine.
CRISPR-Cas14a protein is the smallest RNA-mediated Cas protein, at ~ 400-700aa, half the size of Cas9 protein (~950-1400aa).
CRISPR-Cas14a protein recognizes cleaved single-stranded DNA (ssDNA) with high fidelity. Similar to Cas12a protein, its collateral cleavage activity is activated after specific cleavage of ssDNA, from which the DETECTR assay was developed.
Figure 1. Crispr-cas14a has both cis-and trans-cleavage activities for single-stranded DNA.
(A)Domains of Cas14a,Cas9 and Cas12a proteins.
(B)Cas14a ribonucleoprotein binding to target single-stranded DNA (ssDNA) and its cis-cleavage activity on ssDNA substrate.
(C)Cas14/gRNA/ssDNA activator triplex complex, transcleavage of ssDNA-fluorophore quencher (FQ) reporter gene and emission of fluorescent signal.
Experience
SDS-PAGE Protein expression of Cas14a1
The transformed cells were grown in LB media containing 50μg/ml ampicillin at 37 ℃ and 180 rpm until and OD600 for 0.6. The 10his-MBP- cas14a1 protein expression was induced overnight at 18℃ and 180rpm by adding IPTG to make the final concentration 0.2 M. Cells were centrifuged at 10000 rpm for 10 min at 4 ℃. The pellet was collected and resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM DTT, 0.2 mM PMSF, 5% glycerine, 5 mM β- mercaptoethanol, 10 mM imidazole) and broken by sonication. The temperature was decreased to 4 ℃ and the cell pellet was removed by centrifugation at 8000 rpm and 30 min. The clear supernatant was added into Bio-Scale Mini Nuvia IMAC Ni-Charged (Bio-Rad Laborataries, Inc., USA) and eluted with the linear gradient by increasing in imidazole concentration (from 10 to 300 mM) in 20mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM β-mercaptoethanol buffer. The 10his-MBP-tag was removed with TEV protein at 4 ℃ and overnight incubation. The final solution was replaced to buffer (20 mM Tris-HCl, pH8.0, 100 mM NaCl, 5 mM β-mercaptoethanol) and loaded into heparin column (GE, Hi-Trap) and eluted with linear gradient by increasing in NaCl concentration (from 100 mM to 2M). The fraction containing cas14a1 was collected and stored at - 20 ℃.
There are two bands in the Cas14a1 lane at the approximate size of Cas14a1 (61.9 kDa). This would be consistent with the successful expression of the Cas14a1 protein. Eluent of His×10-tagged Cas14a1 from Heparin-column.
Figure 2. SDS-PAGE analysis of purified Cas14a1 protein, SDS-PAGE gel showing expression of a protein of approximately 61.9 kDa. Consistent with the expected expression of Cas14a1.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1357
Illegal PstI site found at 147
Illegal PstI site found at 313 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1357
Illegal PstI site found at 147
Illegal PstI site found at 313 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1357
Illegal BamHI site found at 951 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1357
Illegal PstI site found at 147
Illegal PstI site found at 313 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1357
Illegal PstI site found at 147
Illegal PstI site found at 313
Illegal AgeI site found at 607 - 1000COMPATIBLE WITH RFC[1000]
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