Design

SFLS iGEMers designed a synthetic device based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system that can accurately identify and eliminate melanoma cells with BRAF mutations. This device includes two parts: a CRISPR/Cas9 system and a regulate system. The CRISPR/Cas9 system includes a single-guide RNA (sgRNA) driven by the U6 promotor and a Cas9 protein driven by the pHEF1A promoter.

CRISPR/Cas9

A sgRNA was designed for mutant BRAF V600E, which specifically binds to mutant BRAF V600E gene in melanoma cells (1). The sgRNA was designed through CRISPR-ERA (http://crispr-era.stanford.edu/). Three sgRNA were designed, finally one was proved to be functional and was adopted.

Cas9 is a DNA endonuclease enzyme guided by sgRNA. When the sgRNA binds to the target mutant BRAF gene, Cas9 protein will recognize the 5’NAG 3’ protospacer adjacent motif (PAM) at the target site and combine with the target DNA sequence upstream of the PAM. As a result, Cas9 induces a DNA double-strand break (DSB) 3bp upstream of the PAM (2). The DSB will be repaired by Non-homologous End Joining (NHEJ), which may cause a deletion, an insertion or a frameshift mutation of the target gene, and result in the loss of its function.

Regulatory System

The regulatory system includes an artificial microRNA (amiRNA) which can specially bind to SAMMSON and an artificial complementary sequence after Cas9. Recently, a long non-coding RNA (lncRNA) SAMMSON was found to be specifically expressed in human melanomas (3). Accordingly, we designed amiRNA complementary to SAMMSON gene. We then added a binding site complementary to the amiRNA after the Cas9 protein. Thus, CRISPR/Cas9 system can be regulated.

As the following animation shows, in normal cells where SAMMSON has no expression, the amiRNA will bind to the binding site after Cas9, and prohibit the transcription of Cas9. In contrast, in melanoma cells, the high expressed SAMMSON binds to the amiRNA and the inhibition of the amiRNA on the CRISPR/Cas9 system will be released, which results in the activation of the CRISPR/Cas9 system cleaving the mutant BRAF V600E gene.

Reference

Hale C R, Majumdar S, Elmore J, et al. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Molecular Cell, 2012, 45(3):292-302.

Koo T, Yoon AR, et al. Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression. Nucleic Acids Res. 2017; 45(13):7897-7908.

Leucci E, Vendramin R, Spinazzi M, et al. Melanoma addiction to the long non-coding RNA SAMMSON. Nature, 2016, 531(7595):518-522.