Team:MIT/CRISPR

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CRISPR - dCas13a

What is CRISPR?

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, which refers to a crucial structure of the immune system of various microorganisms. When a bacterial cell is infected by a recurrent virus, the CRISPR immune system can detect the virus and destroy it before it harms the cell. It accomplishes this by using CRISPR-associated proteins (or Cas proteins), which insert short viral DNA segments called spacers between the short palindromic repeats. The short palindromic repeats and spacers are then constitutively transcribed and processed into short segments called CRISPR RNAs (or crRNAs). The crRNAs bind to trans-activating CRISPR RNA (or tracrRNA)* and a Cas protein, such as Cas-9. This RNA-protein complex then seeks out and cuts the viral sequences that are complementary to the crRNA; thus, destroys the invading virus.

The CRISPR system’s ability to do targeted cutting of nucleotide sequences has proven to be revolutionary for genetic engineering. By utilizing the CRISPR system to target sequences of interest, scientists have been able to do selective knockdown or mutagenesis in nearly every organism the system has been tested. Labs around the whole have developed numerous tools using CRISPR systems that range from diagnosing and treating genetic diseases to regulating gene expression in-vivo [or something more interesting]. However, until very recently, all known CRISPR systems targeted DNA sequences. It was reported in 2016, that a new Cas protein called Cas13a is RNA targeting, opening a whole new realm of possibilities.

*In later improvements, the crRNA and tracrRNA sequences were combined into one RNA sequence called the guide RNA (or gRNA).

What is CRISPR-dCas13a?

Cas13a is a new Cas protein that, unlike previously characterized DNA-targeting Cas proteins such as Cas-9, has demonstrated RNA-guided RNase activity. Originally discovered in Leptrotrichia shahii, Cas13a provides interference against RNA phage. Biochemical characterization has shown that Cas13a cleave ssRNA targets by using catalytic residues in its two HEPN domains. The deactivating these catalytic residues by mutagenesis results in dCas13a, which is unable to cleave RNA; dCas13a is effectively a programmable RNA-binding protein.