Part:BBa_K4140016
Cas12g
Part Description
RNA-guided Among its primary targets are single-stranded RNA substrates, Cas12g is a ribonuclease. Comparing it to other Cas12 proteins that have been found so far, CRISPS-Cas12g selectively detects RNA substrates, making it a potentially useful platform for transcriptome editing and diagnostics. While guided RNAs fold into a "F" shape that is primarily identified by the Rec lobes, a bilobed structure of Cas12g displays a tiny NUC2 and REC2 domain. To change the conformation of the REC and NUC lobes and activate Cas12g, target RNA and crRNA guide combine to form a duplex that is inserted into the cavity in the middle of the structure.
Usage
Cas12g is a RNA-guided protein and differs from other Cas12 proteins by targeting a single strand RNA substrates Making it a potent platform for post transcription modification so we use to control PAH and beta-galactosidase expression as it cleaves the mRNA of PAH and beta-galactosidase at specific site without the need to recognize the PAM Sequence distinguishing it from other Cas proteins preventing the translation PAH and beta-galactosidase just in case of over expression of them or high level of tyrosine and absence of L7Ae as shown in figure 1.
Characterization by mathematical modeling
This model is to simulate the kinetics of the riboswitch (L7Ae with kink turns) that is used in our circuit. The designed circuit is to detect if the increasing substance is either phenylalanine or tyrosine via TyrR. So if phenylalanine level is elevated, L7Ae is formed as it is downstream TyrR that forms a complex via binding with its kink-turn on another circuit; that complex inhibits expression of cas12g, so the circuit will be able to express lacZ alpha (beta-galactosidase) in diagnostic circuit or PAH in the therapeutic circuit. If tyrosine level is elevated with a decreased level of phenylalanine, it activates tyrR inhibitory promoter so no L7Ae would be expressed resulting in cas12g expression to control the circuit as shown figure (4) and graph (1).
Figure (4) illustrates the kinetics of all reactions in riboswitch model
Graph (1) illustrates riboswitch kinetics in which Q represents the condition where L7Ae is expressed and bound to its kink-turns ,therefore inhibiting the expression of cas12g. However, M represents no expression of L7Ae in which cas12g would be expressed to control the circuit if the phenylalanine is absent.
Improvement by directed evolution
After performing mutagenesis prediction of mutational landscape of cas12g 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 (R627W) mutation showed the highest score compared to other mutations. On the contrary, we can see that the (L654F) contributed to the lowest evolutionary fitness to cas12g.As shown in Figure (5)
References
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 313
Illegal PstI site found at 787
Illegal PstI site found at 1729 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 313
Illegal PstI site found at 787
Illegal PstI site found at 1729 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1308
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 313
Illegal PstI site found at 787
Illegal PstI site found at 1729 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 313
Illegal PstI site found at 787
Illegal PstI site found at 1729
Illegal NgoMIV site found at 1230 - 1000COMPATIBLE WITH RFC[1000]
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