Difference between revisions of "Part:BBa K3286010"

Revision as of 11:05, 21 October 2019

dCas9-AcrIIA4 gene circuit

Usage and Biology

CRISPR interference (CRISPRi) makes use of catalytically inactive variants of Cas9 (dCas9) or Cas12a (dCas12a) proteins to suppress gene expression [1]. Identical to their active counterparts, the co-expression of guide RNAs directs the ribonuclease protein (RNP) to its specific DNA target sequence. However, introduction of mutations in the RuvC1 and HNH nuclease domains of Cas9, and the RuvC I and RuvC II domains of Cas12a, cause the Cas protein to lose endonuclease activity, without impeding the DNA binding [2; 3]. This enables the reversible transcriptional inhibition by tightly DNA-bound dCas proteins, contrary to irreversible cleavage by active Cas9 or Cas12a. One way to reverse the effect of dCas-mediated gene repression is through their natural inhibitors, known as anti-CRISPR (Acr) proteins. Acrs are small, phage-derived proteins blocking the natural CRISPR immune system of bacteria [4]. In most cases, they directly interfere with Cas nucleases, blocking binding or cleavage of the target DNA [5]. Therefore, Acrs may represent a powerful tool for the optimization of CRISPR/Cas-based genome editing approaches or the construction of synthetic circuits [6].

Sequence and Features

The dCas9-AcrIIA4 gene circuit mainly consists out of three parts; the AcrIIA4, the dCas9, and the sgRNA expression module. The acr expression is under the control of the L-rhamnose inducible promoter (Prha) and shares a bi-directional terminator with the dCas9 gene (BBa_K3286009). The dCas9 is being expressed via the tetracyclin promoter regulated via the IPTG-inducible lacI/lac operator (Ptet/lac)(BBa_K3286008). The sgRNA (spacer and scaffold) are expressed by the strong constitutive J23119 promoter (BBa_K3286003). The sequence (cds) for terminators, promoters as well as the dCas9 and the sgRNA scaffold were provided by the Laboratory of Microbiology. The sequence of the acrIIA4 gene was retrieved from repositories, such as the iGEM repository or ordered as gene blocks and oligos via Integrated DNA Technologies. The circuit was inserted into the pACYC184 vector with p15a ori and chloramphenicol resistance using High Fidelity Assembly.

Sequence and Features