Part:BBa_K4139009

This part will produce an mRNA transcript which serves as a crRNA for CRISPR Cas13a from Leptotrichia wadei. It includes the T7 Promoter J64997, the RBS B0034, and double terminator B0015.

This crRNA is an RNA structure containing a direct-repeat stem loop, a recognition element for binding with the Lwa-Cas13a protein (see parts BBa_K3738020, BBa_K3738021, BBa_K3738023 and BBa_K3738024). The crRNA also contains a downstream complementary region, designed to base-pair with a target ssRNA sequence. Complex formation of crRNA-Cas13a occurs, and when the target sequence is perfectly paired with the crRNA, activation of the enzyme occurs and subsequent non-discriminate cleavage of collateral ssRNAs (O’Connell., 2019).

This crRNA was designed to bind with an important protein in the synthesis of harmful toxins called microcystins produced by blue-green algae (cyanobacteria) blooms. McyH is a gene in the Mcy gene cluster of Microcystic Aeruginosa and codes for a transporter protein. The protein is comprised of two homodimers, each with a hydrophobic N-terminus domain and C-terminus containing an ATPase domain. Pearson et al., 2004 experimentally examined the impacts of impairing expression of this gene, and combined with bioinformatic data, found that McyH is likely a vital exporter of harmful microcystins and essential in their biosynthetic pathway.

This part is an improved version of the part BBa_K3738022, which we initially designed for our 2021 project. This version of the part is hypothesized to have increased binding affinity and target specificity for the target protein.