CRISPR

CRISPR Technology: The future of genome editing

The realm of genome editing has taken a significant step forward with the recent discovery, called CRISPR technology. CRISPR originates as an immune mechanism used by bacteria to fend off against virus infection. What is fascinating about CRISPR is that bacteria takes a chunk of DNA from virus and store it inside its genome. Then, when the virus infects the bacteria again, the bacteria is able to use the chunk of DNA that it stole as a surveillance tactics. Once it matches the virus DNA, it triggers a cascade of editing of the virus DNA to get rid of the infection.

CRISPR_1

What you see here is the bacteria genome at the bottom. The CRISPR locus (labeled as CRISPR array) is identified by the DNA repeats (black diamonds), with specific virus DNA (multicolor boxes) spaced between these repeats. Upstream of this CRISPR locus is the Cas locus, which consists of important enzymes and proteins that assemble and facilitate the excision.

CRISPR stands for clustered regularly interspaced short palindromic repeats). Inside the CRISPR locus, you will see DNA repeats intercalated with specific DNA sequences (in the diagram it says spacer). These DNA sequences are sequences that the bacteria acquired from previous round of infection. After transcription, these DNA repeats are transcribed into RNA that carries hairpin structures. These RNA are called crRNA. These crRNA can then be used to target unwanted pathogens to degradation.  Upstream of this CRISPR locus, you will be able to find cas genes.

CRISPR_2

CRISPR locus generates hairpin RNA, crRNA. Cas locus generates Cas proteins.

Depending on what types of CRISPR, some or just one Cas genes are required for assembling and cutting the target DNA.

Since a lot of inventions are evolved around the type II CRISPR system/Cas9, I will focus on the type II CRISPR system that requires a large multifunctional protein to destroy invading DNA and a trans-activating crRNA (tracrRNA).  After crRNA binds to the target DNA, tracrRNA binds to crRNA and recognized by Cas9 and then cleaved by RNase III

CRISPR_3

crRNA identifies target DNA. tracrRNA then binds to crRNA, which triggers the Cas9 protein to latch on. This triggers RNAIII in forming a double stranded break in the target DNA

From this knowledge, scientists have derived a new strategy in genome editing. They combined the fundamentals of crRNA and tracrRNA. So now, you can customize the stretch of crRNA and then add on the chunk of tracrRNA that Cas9 requires to activate the excision cascade (see below).

The crRNA can target the gene that you want to target. Though you still have to express Cas9, in order to facilitate the genome editing. 

CRISPR_4

Q&A: What do superantigens do?

Superantigens activate 5-20% of T cells with massive cytokine release (i.e. IL-1, TNF-alpha, interferon, etc). This activation is not specific to any antigen. Superantigens interact with MHC and T cell receptor at sites distinct from normal antigen binding and antigen presenting sites. This interaction leads to T cell activation without internalizing the superantigens.