Difference between revisions of "Part:BBa K4223003"

Revision as of 12:40, 21 September 2022

Transcription system of target RNA for LbCas13a

Cas14a 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 Cas14a recognized and cleaved the target RNA.

Experience

Cas14a system
In order to provide data support for false positives, we introduced 13 DNA sequences into Cas14a detection system, including 1 Target and 12 DNA 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 DNA and Target

Through the recognition and cleavage of the artificially designed RNA by Cas14a protein, the trans-cleavage activity was displayed, 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 M20 and M7 were significantly greater than the target sequences. 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 Cas14a protein for each sequence (revealed by relative fluorescence unit RFU)

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

Centrifuge tube system

30uL /cell
Cas14a

Reaction Buffer (20 mM tris-hcl, 20 mM NaCl, pH 9.0)

15 uL: Cas14a （final 500nM） &sgRNA (final 500nM) in Reaction buffer ;
6 uL: Mg ion ( final 10mM) & FQ (final 400nM in reaction buffer);
9 uL: T or Mx & Clamps in reaction buffer.

Sequence and Features