Part:BBa_K4223003

Transcription system of target RNA for LbCas13a

Cas13a tolerates one or two mismatches between sgRNA and the target sequence, which will result in a greatly reduced cleavage efficiency of Cas14a protein, and this wrong cleavage will also lead to "false positives" in the detection.
In order to confirm the occurrence of false positives, Hainanu-China designed a series of sequences with single nucleotide polymorphisms (SNPS) based on the target RNA, which were detected by fluorescence reporter.
It should be noted that fluorescence was generated by trans-cleavage of ssRNA cleavage Reporter after Cas13a recognized and cleaved the target RNA.

Experience

Cas14a system
In order to provide data support for false positives, we introduced 13 RNA sequences into Cas13a detection system, including 1 Target RNA and 12 RNA mutated sequences containing odd locus single base mutations. The specific mutation sites are shown in Figure 1.


Figure 1. Single nucleotide polymorphisms(SNPS) at odd loci in 12 different RNA and Target RNA

Through the recognition and cleavage of the artificially designed RNA by Cas13a protein, the trans-cleavage activity was activated, and the resulting fluorescence signal was revealed as Relative fluorescence Unit (RFU). We could observe that each detected sequence showed different fluorescence signal intensities, and the signal intensities of RM8 RM15 and RM19 were significantly greater than the target RNA. This indicates that false positives do exist, and different mutation sites have different effects on the specificity of the detection.

Figure 2. Detection results of Cas13a protein for each sequence (revealed by relative fluorescence unit RFU)

Figure 3. Schematic representation of the Cas14a-sgRNA-Target complex

Centrifuge tube system

(2x) buffer: 20 mM tris-hcl, KCl 100 mM, pH=8.2.

20 uL /cell：

10uL buffer(2x);
0.8uL 500nM Cas13a;
0.1uL 5uM crRNA;
0.67uL 150mM Mg2+(ca. 5mM Mg2+ final.)

37 ℃ Incubation 10-15min, add probe 0.6uL（10uM） FQ ;
（12.17 uL in total ca. 12 uL)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 100
  
• 1000
  COMPATIBLE WITH RFC[1000]