Part:BBa_K3743015
L7Ae (Cas12g) linked by Gly Ser Linker
Part Description
CRISPR-Cas12g protein belongs to the RNase protein family. This family is a single-component programmable RNase with functions in RNA processing and programmed cleavage of RNA. It is linked by Gly Ser Linker and "L7AE" which belongs to the family of proteins that bind k-turns in RNA to stabilize tightly kinked versions. RNA-protein complexes played a key role in translation, splicing, and modification of site-specific RNA. When cas12g binds to mRNA from the cancerous environment, L7Ae is consumed, disinhibiting the circuit, and, therefore, transcripting more copies.
Usage
The Gly Ser linker is utilised to conjugate it with the L7Ae protein (which inhibits transcription by binding to its kink-turn). The mechanism simply identifies and attaches to mRNA in the cancerous environment, causing the L7Ae protein to be consumed. As a result of not binding to kink-turn, the inhibitory action on transcription is inhibited, resulting in transcription activation and a rise in vaccine yield. As a result, it is a cell-specific design that binds to PD-L1 mRNA, which plays an immune evasion role in cancerous environments, particularly TLCs.
Literature Characterization
A Cas12g–sgRNA–target RNA ternary complex was assembled by incubating a catalytically inactive Cas12g (E655A) with the sgRNA and a 24-nucleotide target RNA in order to obtain insight into target RNA recognition. By using cryo-EM, we were able to determine the structure with a 4.8 resolution. As shown in figure (1). The nuclease activity was abolished when REC1220–354 was deleted, showing that it plays a crucial role in substrate cleavage (Figure 2). The positively charged residues in the REC1220–354 region are obviously involved in the recognition of the crRNA–target RNA duplex. Mutation of six positively charged residues within the first 20 amino acids of this region (K221, K223, R224, R226, R232, and K237) had a comparable effect on RNA cleavage efficiency as deletion of REC1220–354 in support of this statement.(1)
Characterization Of Mutational Landscape
After performing mutagenesis prediction of mutational landscape of L7Ae 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 (G108S) mutation showed the highest score compared to other mutations. On the contrary, we can see that the (G108K) contributed to the lowest evolutionary fitness to L7Ae. As shown in Figure (3). Which was used to also charectrize and improve part BBa_K2100068
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 (4)
References
1.Li, Z., Zhang, H., Xiao, R., Han, R., & Chang, L. (2021). Cryo-EM structure of the RNA-guided ribonuclease Cas12g. Nature Chemical Biology, 17(4), 387–393.
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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