Coding

Part:BBa_K3743005

Designed by: Ahmed Gamal Mohamed Mattar   Group: iGEM21_AFCM-Egypt   (2021-09-21)


dCas13

Part Description

Cas13 proteins recently identified RNA-guided and RNA-targeting RNase protein family, including Cas13a, Cas13b, Cas13c, and Cas13d, which are single-component programmable RNases with functions in RNA processing and programmed RNA cleavage it also allows robust real-time imaging and tracking of RNAs in live cells,even when utilizing single guide RNAs of 20 to 27 nucleotides. Cas13b was found to be the most stable and robust of the four kinds in mammalian cells for RNA knockdown and editing.

Usage

It's a deactivated Cas13 that's linked to the L7Ae protein (which inhibits transcription by binding to its kink-turn) with a Gly Ser linker. The mechanism simply detects and binds to mRNA in the cancerous environment, causing the L7Ae protein to be consumed. As a result of not binding to kink-turn, the inhibitory effect on transcription is inhibited, resulting in transcription stimulation and an increase in vaccine yield. That is why, it is a cell specific design by binding to mRNA of PD-L1 which has an immune evasion role in the cancerous environment especially TLCs.






Literature Characterization

A study tried to determine whether the dPspCas13b fusion proteins could promote CRISPR-Cas13-mediated cleavage and polyadenylation of the sfGFPapa reporter mRNA, so they designed a crRNA to target an intronic sequence which was downstream of sfGFP in the sfGFPapa reporter as shown in figure (1.a) after doing the experiment in live assays they found no fluorescence signal in cells when using a non-targeting crRNA in comparison to those with intron targeting crRNA that expressed sgfp resulting in detectable green fluorescent light after 24 hrs as shown in figure (1.b). (1)

Figure 1.CRISPR-Cas13-mediated Cleavage and Polyadenylation of a Reporter mRNA in Mammalian Cells.(a)Shows a diagram with the implemented targeting strategy(b) Shows expression of the dPspCas13b-NUDT21 protein and intronic targeting crRNA resulting in green fluorescent cells after 24 hours.




















Characterization Of Mutational Landscape

After performing mutagenesis prediction of mutational landscape of dcas13 and tested the effect of these mutations on the evolutionary fitness of the protein after generating multiple sequence alignment of the protein sequence and predict mutational landscapes. As shown in the chart, the (A472R) mutation showed the highest score compared to other mutations. On the contrary, we can see that the (A477K) contributed to the lowest evolutionary fitness to dcas13. As shown in Figure (2)

Figure 2.shows the positive fit mutants upon saturation mutagenesis prediction of mutational landscape of dcas13



















References

1.Anderson, K. M., Poosala, P., Lindley, S. R., & Anderson, D. M. (2019). Targeted cleavage and polyadenylation of RNA by CRISPR-Cas13. BioRxiv, 531111. 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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



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