Difference between revisions of "Part:BBa K4586004"

Revision as of 17:47, 3 October 2023

Cas12k

Part Description

This part codes for the type V-K CRISPR-Cas system effector protein Cas12k, that is characterized by naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA or genome editing, Its protospacer adjacent motif (PAM) sequence is GGTT. that is essential for activating the enzymatic activity of the Cas protein, and the guide RNA is designed based on the PAM sequence site corresponding to the target gene.

Usage

This part is implemented in our design to be the main element of our therapeutic agent (Cargo) to knock-out the BAFF-R gene apoptosis, which is essential for B-cell survival and maintenance, to cause apoptosis in the autoreactive B-cells that secrete Anti-citrullinated Protein Antibody (ACPA). Its guide RNA is designed to drive the protein portion of the CRISPR Cas system Cas12k to the BAFF-R gene as shown in figure 1.

Figure 1: This figure illustrates the activity of the Cas12k protein into the target autoreactive B-cells through knocking down the B-cell activating factor receptor (BAFF-R) gene that is responsible for B-cells survival and proliferation leading to their apoptosis.

Literature Characterization

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 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.

References

Xiao, R., Wang, S., Han, R., Li, Z., Gabel, C., Mukherjee, I. A., & Chang, L. (2021). Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition. Molecular Cell, 81(21), 4457-4466. Sequence and Features