Part:BBa_K4223020
A protein expression system of Cas13a
Mechanism of action of Cas13a: After the CRISPR-Cas13 system binds to crRNA and substrate to form a ternary complex, Cas13 protein is activated, at which time Cas13 protein can cut not only substrate RNA but also any single-stranded RNA that is free within the environment. cas13a protein and Cas13b protein are RNA enzymes, and Cas13a and Cas13b based Cas13b assays require the transcription step of RNA polymerase.
Cas13a, which requires only 24 bases of crRNA, interacts with the Cas13a molecule through a uracil-rich stem-loop structure and facilitates cleavage of the target through a series of conformational changes in Cas13a. Like Cas9, Cas13a tolerates a single mismatch between the crRNA and the target sequence, although if 2 mismatches are present, then the efficiency of cleavage is greatly reduced. Its PFS sequence (equivalent to the PAM sequence) is located at the 3' end of the spacer region and consists of A, U or C bases.
Another special feature of Cas13a is that once Cas13a recognizes and cleaves the RNA target specified by the crRNA sequence, it enters an enzymatic "activated" state where it will bind and cleave other RNAs regardless of their homology to crRNA or the presence of PFS. This is very different from Cas9. In the case of bacteria, this property makes sense. If a bacterium is infected by a phage, it is able to activate programmed cell death or a dormant state to limit the spread of the infection throughout the population.
Like Cas13a, Cas13b requires only a single guide RNA to find its target and is codable from a genetic standpoint. In addition, Cas13b is capable of targeting multiple RNA transcripts simultaneously. However, Cas13b also has some unique features that suggest it is distinct from Cas13a. These features make Cas13b more suitable for fine-tuning gene function
Experience
Cas13a 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.
_at_odd_loci_in_12_different_RNA_and_Target_RNA.png)
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.
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Figure 2. Detection results of Cas13a protein for each sequence (revealed by relative fluorescence unit RFU)
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1597
Illegal PstI site found at 2452
Illegal PstI site found at 3391 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4019
Illegal PstI site found at 1597
Illegal PstI site found at 2452
Illegal PstI site found at 3391 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 338
Illegal BglII site found at 878
Illegal BglII site found at 1814
Illegal BglII site found at 2024
Illegal BglII site found at 2078
Illegal BglII site found at 2309
Illegal BglII site found at 2582 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1597
Illegal PstI site found at 2452
Illegal PstI site found at 3391 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1597
Illegal PstI site found at 2452
Illegal PstI site found at 3391
Illegal NgoMIV site found at 2530
Illegal NgoMIV site found at 3166 - 1000COMPATIBLE WITH RFC[1000]
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