Casilio® Mammalian Expression Vectors

The Casilio mammalian expression vectors were developed with the Casilio system, an approach that has distinct advantages in multiplexing and multimerization, in comparison to conventional CRISPR/Cas9 usage. Casilio has the unique ability to simultaneously regulate multiple target genes in a cell, providing a more powerful way of studying genes inside living organisms. Discover the flexibility and compatibility of Casilio vectors for gene editing, transcription regulation, and DNA-binding fluorescent protein-based detection.

What is the Casilio System?

Casilio is a hybrid system combining CRISPR/dCas9 and the Pumilio RNA-binding domain (fig.1). The Pumilio/PUF RNA binding domain consists of eight sequence repeats, each capable of recognizing a single RNA base. Within each repeat, specific residues can be altered to facilitate recognition of each of the four RNA bases. In contrast to the conventional approach of directly attaching effector domains to dCas9 in CRISPR/dCas9-based enzymes, effector proteins are repositioned. This was achieved by modifying the sgRNA (sgRNA-PBS), which contains varying numbers of 8-mer Pumilio binding sites (PBS) that can then recruit effector domains fused to cognate PUF domains.

Required Components to Perform Casilio Experiments (using Sp-dCas9)

• Sp-dCas9: nuclease-deficient S. pyogenes Cas9 with D10A and H840A mutations
• Casilio dCas9 Mammalian Expression Vector ( ABIN7529448 ): Transient expression vector for dCas9
• sgRNA-PBS: A sgRNA appended with multiple copies of Pumilio binding sites (PBS).
• Casilio modules : A fusion protein consisting of effector domain(s) fused with a PUF domain programmed to recognize a specific 8-mer PBS.

What are the Advantages of the Casilio System?

The Casilio system overcomes several downfalls of classic dCas9 vectors. dCas9 fusions cannot be co-introduced with sgRNAs to regulate two set of genes differently since both fusions will bind to both set of genes directed by both sets of sgRNAs. Additionally, often more than one molecule of effector proteins is desired to achieve sufficient biological activity. Existing solutions such as modified sgRNA extended with structural aptamers show inhibitory effects when used in higher numbers. Key advantages of the RNA-aptamer based approach of the Casilio system are:

• Multiplexing: Different sgRNA-PBS allow simultaneous recruitment of various effectors at different target sites.
• Flexibility: No need for specific Cas9 fusions proteins depending on the effector; sgRNA-PBS and Casilio-modules vary.
• Compatibility: No need to change existing work-flows and SOPs.

Casilio modules are orthogonal, allowing simultaneous and independent activation and repression of different genes (Fig. 2). Additionally, Casilio can multiplex and multimerize effectors and potentially enable assembly of stoichiometrically defined protein complexes (Fig. 3). This makes the Casilio system an ideal option for transcription regulation, fluorescent protein-based detection as well as gene editing. Explore our available vectors for the Casilio system below.