Biotech Breakthrough: The CRISPR/Cas System

 

By John McLaughlin

In the last few years, a huge amount of excitement has grown over the CRISPR/Cas system and its use in targeted genome editing; this acronym derives from Clustered Regularly Interspaced Short Palindromic Repeats and their CRISPR-associated genes (Cas). CRISPR loci, which are found in many species of bacteria and most archae, have been collectively described as an RNA-based “immune system,” because of their ability to recognize and destroy foreign phage and plasmid DNA.

 

Although the acronym was first coined in a 2002 paper, CRISPR has only recently been exploited as a research tool. How does the system work and what is its use in the lab? There are at least three distinct types of CRISPR system. A typical “type II” CRISPR locus consists of several protein-coding Cas genes adjacent to an array of direct repeat and spacer sequences. The direct repeats are usually palindromic and conserved, in contrast to the much more variable spacers; these repeat-spacer sequences are transcribed as one unit and then processed into short CRISPR-RNAs (crRNAs).  A 2007 Science article demonstrated that a bacterial population could acquire resistance to phage infection by incorporating DNA fragments from the invading phage genome into a CRISPR locus, in the form of new spacer sequences. The newly acquired spacers are then transcribed and processed into crRNAs, associate with a trans-activating RNA (tracRNA) and Cas protein, and are eventually guided to a homologous DNA sequence to catalyze a double-stranded break.

 

The CRISPR system can be flexibly “reprogrammed” by designing custom chimeric RNAs (chiRNA), which serve the function of both crRNA and tracRNA in one molecule. By co-expressing a “designer” chiRNA with a Cas protein, a targeted and specific DNA break can be created in the genome; after providing an exogenous DNA template to help repair the break, customized knock-ins or knock-outs can be generated. Judging from the rapid technical advances made in the last few years, the system promises to be an efficient and high-throughput format for genome editing. To date, knock-outs have been created in a variety of organisms including rats, flies, and human cells.

 

CRISPR/Cas technology has attracted scientific attention as well as commercial interests. In November 2014, biologists Jennifer Doudna and Emmanuelle Charpentier were honored as co-recipients of the 2015 Breakthrough Prize in the Life Sciences, for their work in dissecting the mechanism of CRISPR’s sequence-specific DNA cleavage. According to its proponents, the possible applications of the CRISPR system seem almost limitless. CRISPR Therapeutics, a recently formed company dedicated to translating the technology into genetic disease therapies, has raised 25 million dollars from new investors. And just last month, the pharmaceutical company Novartis began collaborations with Intellia Therapeutics and Caribou Biosciences in order to pursue new therapeutics using CRISPR/Cas.

 

A technology as potentially lucrative as this one does not develop without controversy. MIT Technology Review recently reported on the competing startup companies aiming to exploit CRISPR technology, and the ensuing battles over intellectual property rights in different organisms. In fact, last year the Broad Institute and MIT were awarded a patent which covers the use of CRISPR genome-editing technology in eukaryotes. Feng Zhang, who is listed as Inventor on the patent, and his lab at MIT were the first to publish on CRISPR’s functionality in human cells.

 

In a few years, this exciting technology may be a commonplace fixture of the biology lab. Only time will tell if the CRISPR craze produces the amazing breakthroughs that scientists, and the general public, are eagerly awaiting.