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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1357
  Illegal PstI site found at 147
  Illegal PstI site found at 313
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1357
  Illegal PstI site found at 147
  Illegal PstI site found at 313
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1357
  Illegal BamHI site found at 951
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1357
  Illegal PstI site found at 147
  Illegal PstI site found at 313
  
• 25
  INCOMPATIBLE 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
  
• 1000
  COMPATIBLE WITH RFC[1000]