Part:BBa_K4139004

When combined into a BioBrick with a promoter and terminator (but without an RBS), this part will produce an mRNA transcript which serves as a crRNA for CRISPR Cas13a from Leptotrichia buccalis (called Lbu Cas13a).

This crRNA is an RNA structure containing a direct-repeat stem loop, a recognition element for binding with the Lbu-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. It was designed with the help of the RNAfold software tool, which allowed us to locate a target sequence that met the ideal design criteria of being close to the 5' end of the sequence, being a relatively unfolded region in the mRNA secondary structure, and having a high probability of remaining single-stranded.