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On the road to harnessing CRISPR gene editing to treat cancer


 

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CRISPR technology, a simple, yet incredibly powerful tool for genetic engineering, is not only allowing cancer researchers to screen for drug targets more efficiently, but is also opening the door for direct cancer treatment through gene interference or activation.

“The pace at which this technology is developing is astounding and almost every cancer research lab is now using some version of it in their studies,” Dr. Scott A. Armstrong, director of Memorial Sloan Kettering Leukemia Center, New York, said in an interview.

Dr. Scott Armstrong

Dr. Scott Armstrong

Ancient defense mechanism

While the term CRISPR is now synonymous with the editing of human genes, it actually refers to a sort of primitive immune system used by bacteria for billions of years. CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, but the complex terminology belies an elegantly simple mechanism by which bacterial cells destroy invading pathogens.

The bacterial genome contains regions with short repetitive stretches of DNA that are separated by spacers. Researchers made the startling discovery that the spacers are often composed of bits of foreign DNA and it transpired that bacteria use it as a molecular memory of prior infection.

When the same pathogen is encountered again, the stretches of repeats and spacers are transcribed to form CRISPR RNAs (crRNA). Together with a transactivating RNA (tracrRNA), it forms a kind of GPS system for a series of CRISPR-associated (Cas) proteins that function like molecular scissors, destroying the target DNA sequence in the invader’s genome.

There are three CRISPR systems – type I, II, and III – that are associated with different sets of Cas proteins and each employ unique methods for achieving the same ultimate function. The type II system that pairs with Cas9 has received the most attention.

Cut and paste gene editing

The discovery that CRISPR could be exploited as a tool for genetic manipulation in mammalian cells sparked a revolution in the genome editing field. “The CRISPR-Cas9 system has been adapted to specifically edit the genomes of mammalian cells, allowing one to make targeted changes to almost any gene,” Dr. Armstrong said.

The use of CRISPR-Cas9 as a genome editing tool is simplified by joining the crRNA and tracrRNA together so they are transcribed in a single guide RNA (gRNA). The GPS coordinates of the gRNA can be preprogrammed to target a gene of interest, specifically directing the co-transcribed Cas9 protein to cut at that location, and introducing a double-strand break (DSB) in the DNA. Cells employ a number of different mechanisms to repair DSBs and these can then be exploited for genome editing purposes, allowing researchers to introduce changes to the DNA as it is repaired.

The CRISPR-Cas9 system excels in its simplicity – allowing alterations to be made to the genome much more easily, quickly, and cheaply than ever before, plagued by far fewer off-target effects. It also allows researchers to examine the function of multiple genes at once, where before they were mostly limited to a single gene.

Dr. Tyler Jacks Alisha Siegel

Dr. Tyler Jacks

“Cancer genomics has identified a large number of genes that are mutated in human cancer,” Tyler Jacks, Ph.D., director of the Koch Institute for Integrative Cancer Research at MIT, Boston, said in an interview. “CRISPR allows us to study these genes in cancer cells and in whole animals much more efficiently than the methods that were in use just a few years ago.”

But the potential of the CRISPR-Cas9 system doesn’t stop there. “At the moment, a particularly exciting application is the use of this approach to inactive genes in very specific fashion to assess the function of a given protein in a cancer cell, which should speed the identification of proteins that are important for cancer cells and thus potentially aid drug discovery efforts,” Dr. Armstrong said.

CRISPR at AACR

The latest developments in the use of the CRISPR-Cas9 system were highlighted at the annual meeting of the American Association of Cancer Research. Dr. David Sabatini, professor of biology at MIT, Boston, described his own lab’s method for using CRISPR-Cas9 to seek out the essential genes involved in different types of cancers. In a study recently published in Science, he and his colleagues employed this method in chronic myelogenous leukemia and Burkitt’s lymphoma cell lines. The gRNA library targeted just over 18,000 genes and roughly 10% of these proved to be essential. Mostly, these genes were linked to key cellular processes (Science 2015;350[6264]:1096-1101).

Dr. Christopher Vakoc of Cold Spring Harbor (N.Y.) Laboratory, presented a slightly different kind of CRISPR screen for drug targets. Most commonly, CRISPR introduces edits at the start of the gene, which may or may not change the DNA enough to produce a nonfunctional protein. Dr. Vakoc’s lab has developed a system that instead edits functional protein domains, which present ideal drug targets. Mutated domains can be identified that are essential for cancer cell survival and small molecule inhibitors designed that bind to them to kill cancer cells.

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