Part:BBa_K4586025
Cargo (guide RNA - switch- Cas12k- MCP/ADAR)
Description and Usage
The first component of Cargo is the CRISPR system, where we uses guide RNA to direct our Cas12k toward the BAFF-R gene in auto-reactive B-cells, and the second component is the DART V ADAR switch, where we uses ADAR enzyme to convert Adenosine into Inosine, causing a change in the switch condition from off to on status, but ADAR enzyme works only in the presence of complementary mRNA in the target cell to the sensor within the switch that contains the stop codon (UAG) of auto-reactive B-cells. This stop codon contains a mismatched Adenosine (A)group with the complementary mRNA; this group is hybridized by ADAR enzyme activity and converts to an Inosine group (I); thus, the stop codon sequence is disturbed in this condition and our cargo will be translated as shown in figure 1.
Figure 1: This figure illustrates the design of our biological circuit expressing our therapeutic agent under the control of the VP64 transcription module that regulates the activity of the ZF21.16minCMV promoter.
Figure 2: This figure illustrate the activity of our DART V ADAR tissue specific switch that is designed to be in the on state after recognition of the autoreactive B-cells,this recognition based on mismatched base editing in the level of transcribed RNA that is mediated through ADAR enzyme activity.
literature charactrization of MCP/ADAR
The study tested the action of sensors containing MS2 hairpins without ADAR, with ADAR p150, or with MCP-ADAR2dd.
Off-state refers to mNeonGreen expression in the absence of iRFP720 trigger mRNA, while on-state refers to mNeonGreen expression in the presence of iRFP720 trigger mRNA. They found that constitutive expression of MCP-ADAR causes an increase in sensor activation in the absence of the trigger.
Literature Characterization of cas12K
In order to purify Cas12k, it was expressed in Escherichia coli.Cas12k's capacity to catalyze DNA transposition was tested using gel electrophoresis. To ascertain the background level of transposition, a negative control response devoid of Cas12k was used.
The graph displays the average and standard deviation of three independent replicates from a representative experiment. The level of DNA transposition was markedly elevated by the addition of Cas12k.
Characterization of cas12K By Mutational Landscape
In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (A8C,F13S) shows the highest epistatic fitness, while the lowest score was associated with the mutation (R536E).
Figure . An illustration of the effects of different mutations on the Epistatic Fitness of cas12K.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 5988
Illegal AgeI site found at 6100 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1456
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