Part:BBa_K4053001

LBCas12a-RVRR

This is an improvement of part BBa_K2927005, LbCas12a

An evolved mutant of LbCas12a, LbCas12a-RVRR with mutations at G532R, K538V, Y542R and K595R.

The LbCas12a-RVRR has a more varied PAM, achieved by introducing the mutations at the analogous positions of the RVR and RR variants of LbCas12a. As the trans-cleavage activity of the LbCas12a-RVRR was not characterised, we decided to test its activity to detect point mutation. We detected the same target (BRAF V600E) and we managed to further improve the sensitivity for the same target mutation detection. We made several mutations for WT-LbCas12a plasmid and managed to purify the LbCas12a-RVRR. We also tested it in the FQ Assay and found out that it can better differentiate mutant: wild-type template, hence providing higher sensitivity for snip detection.

Results

After complexing WT-LbCas12a and LbCas12a-RVRR to the gRNAs, we tested their detection performance against BRAF-V600E by using the FQ (Fluorophore-Quencher) reaction and recorded the fluorescence signal.

The figure above shows the signal of ratio:mutant (BRAF-V600:BRAF-E600) for WT-LbCas12a (left) and LbCas12a-RVRR (right) recorded at the 30th minute in 37°C in the FQ reaction.

The higher signal from LbCas12a-RVRR shows that LbCas12a-RVRR can differentiate BRAF-V600E better than WT-LbCas12a. Therefore, our experiment has proven that LbCas12a-RVRR has a higher sensitivity towards mutation in the BRAF gene.

References

Tóth, E., Varga, É., Kulcsár, P. I., Kocsis-Jutka, V., Krausz, S. L., Nyeste, A., Welker, Z., Huszár, K., Ligeti, Z., Tálas, A., & Welker, E. (2020). Improved LBCAS12A variants with altered pam specificities further broaden the genome targeting range of Cas12a nucleases. Nucleic Acids Research, 48(7), 3722–3733. https://doi.org/10.1093/nar/gkaa110

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1201
  Illegal PstI site found at 1699
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1201
  Illegal PstI site found at 1699
  Illegal NotI site found at 3745
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3123
  Illegal XhoI site found at 3754
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1201
  Illegal PstI site found at 1699
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1201
  Illegal PstI site found at 1699
  Illegal NgoMIV site found at 3699
  Illegal NgoMIV site found at 3718
  Illegal AgeI site found at 3427
  
• 1000
  COMPATIBLE WITH RFC[1000]