Part:BBa_K4204001

Coding sequence of Cas12b

Cas12b, also previously known as C2c1. It is a thermophilic RNA-guided endonuclease that is part of the HOLMES (one-HOur Low-cost Multipurpose highly Efficient System). It exhibits cis-cleavage activity on target double-stranded DNA (dsDNA) and trans-cleavage activity on ssDNA. In the HOLMESv2 system, the latter is employed to cleave the probe to emit fluorescence. The trans-cleavage activity is protospacer adjacent motif (PAM) dependent. However, it requires binding with sgRNA which recognizes the PAM sequence on the target DNA for cleavage. After binding and targeting DNA, Cas12b will trans-cleave the probe and hence emit detectable fluorescence.

Contribution of Squirrel-CHN-II 2024

Point mutation of C2c1 (AacCas12b) improves trans-cleavage efficiency.

During our review of literature, we found that K396A/G478A of AapCas12b can improve its trans-cleavage efficiency (Fig.1)[1], but there is no protein structure of AapCas12b.

Fig.1 Trans-cleavage results for AapCas12b single point mutations and double point mutations[1].
A, Illustration of residues interacting with PAM and DNA in AacCas12b. The structure of AapCas12b is highly conserved with AacCas12b. B and C, Fluorescence value of AapCas12b-WT and mutants in LAMP-mediated one step testing. Single mutation (c) and double mutations (d) were tested.

Through the protein comparative analysis software, we found that AapCas12b is highly conserved with AacCas12b, and the sequence similarity between them is more than 90% (Fig.2).

Fig.2, Comparison of amino acid similarity between AapCas12b and AacCas12b (partial) where red boxes indicate mutation sites.

Therefore, we predicted the protein structure of AapCas12b using Alphfold2 (Fig. 3A) and compared AapCas12b and AacCas12b (PDB: 5U34) structurally, and found that both were highly conserved at the point of K396/G478 (Fig. 3A, B), therefore, we concluded that the K396A/G478A mutations can also be applied to AacCas12b.

Fig. 3, Structural comparison of AapCas12b and AacCas12b.
A, Predicting the structure of AapCas12b using AlphaFold2. B and C, Comparative results of AapCas12b (red) and AacCas12b (green). The results show that both are highly conserved in K396/G478.

Reference:
[1]Tong, X., Zhang, K., Han, Y. et al. Fast and sensitive CRISPR detection by minimized interference of target amplification. Nat Chem Biol 20, 885–893 (2024).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1060
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3234
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 88
  Illegal NgoMIV site found at 421
  Illegal NgoMIV site found at 457
  Illegal NgoMIV site found at 2732
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2539
  Illegal SapI.rc site found at 3213