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CRISPR wins the Kavli

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Every once in a while, a scientific advancement pushes the boundaries of human capabilities in ways that seem nothing short of magical.

For their invention of CRISPR-Cas9, Emmanuelle Charpentier, Jennifer A. Doudna, and Virginijus Šikšnys recently received the 2018 Kavli Prize in Nanoscience. Certainly, no (ahem) small feat. But what is CRISPR-Cas9 and how does it work? Today, we will explore a bit of the history behind this revolutionary technology and why is it creating such a large stir in the scientific and lay communities alike.

Nanosurgery for the Genome

First awarded in 2008, the Kavli Prize in Nanoscience recognizes outstanding contributions to study of the absurdly small. CRISPR-Cas9, a new technology designed to manipulate life at the genomic level, is an exciting example of how nanoscience may be applied to solving large problems. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, which is just a fancy way of saying short nucleotide segments that read the same forward and backwards. Cas9, standing for CRISPR associated protein 9, is an RNA-guided DNA endonuclease. Simply put, the CRISPR-Cas9 system is like a pair of scissors that allow researchers to cut DNA at precise locations to add or delete genetic material.

Co-opting Bacterial Immunity

CRISPR-Cas9 is derived from the mechanism bacteria use to guard against viral infection. Consecutive DNA repeats separated by non-repetitive spacer sequences were first observed in E. coli in 1987 (1). Jansen and colleagues coined the term CRISPR to describe them, and hypothesized a functional relationship with a set of CRISPR-adjacent genes (cas) encoding proteins with helicase and nuclease motifs (2). Researchers later showed that CRISPR spacer sequences were comprised of foreign genetic material, incorporated following bacteriophage infection. Eventually, it was discovered that a complex consisting of Cas proteins and multiple spacer-derived RNA transcripts could provide E. coli immunity against a DNA virus (3). In essence, it was determined that the CRISPR-Cas system amounts to a form of adaptive immunity; bacteria hold onto pieces of viral DNA to "remember" past infections. If the bacterium encounters the virus again, the transcribed RNA is used by Cas as a guide to cleave the foreign DNA.

A new approach to an old idea

Emmanuelle Charpentier, Jennifer A. Doudna, and Virginijus Šikšnys simplified this system around one of these Cas proteins, Cas9 (4,5). They showed that a single RNA guide strand, combined with the Cas9 protein, could be reprogrammed to cleave DNA at a desired site. Non-homologous end joining or homology directed repair in the presence of a DNA repair template produces deletions or insertions. In the lab, researchers can now create short guide sequences of RNA (sgRNA) directed against a target sequence. The sgRNA is then joined with Cas9 in a plasmid and transfected into cells by viral or non-viral means. Aside from knockouts or knock-ins, dead Cas9 (dCas9) lacking nuclease activity can be used to induce gene expression when fused with a transcriptional activator. As a practical laboratory procedure, genomic editing with engineered nucleases has been around for a while. Prior to CRISPR-Cas9, zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) were the predominant genomic editing technologies. Both ZFN and TALEN can create site-specific double stranded breaks, but require the difficult and time-consuming process of creating a custom protein for each target sequence. The large advantage of CRISPR-Cas9 is that designing an sgRNA sequence is much more straightforward, and adding more than one sequence makes it easily multiplexable (6).

Limitless possibilities

The excitement generated by CRISPR-Cas9 is understandable, given the multitude of ways mankind stands to benefit from this technology. The applications for treating and eliminating disease are staggering. HIV, a virus that integrates itself into the host genome, is a particularly attractive target for a potential CRISPR-based therapy. Kaminski et al. succeeded in excising portions of integrated HIV-1 DNA in living mice, and again in latently infected CD4+ T lymphocytes from HIV patients (7,8). In the area of cancer medicine, several clinical trials are currently underway investigating PD-1 knockout T cells and CRISPR-Cas9 edited anti-CD19 CAR-T cells (9).

CRISPR-Cas9 has amazing potential not only to treat disease, but to increase crop yields, reduce pesticide use, produce biofuels, or even produce wooly mammoth/elephant hybrids. Mammophants aside, CRISPR-Cas9 represents a valuable tool for research as we even use it at BioLegend to validate knockdown/knockout targets. CRISPR-Cas9 is a promising source of new medicines and an exciting look into what the future may hold.

Image courtesy of Dzu-Doodles.

References:
  1. Ishino Y, et al. 1987. Journal of Bacteriology. 169(12): 5429-33.
  2. Jansen R, et al. 2002. Molecular Microbiology. 43(6): 1565-75.
  3. Brouns S, et al. 2008. Science. 321(5891): 960-964.
  4. Jinek M, et al. 2012. Science. 337(6096): 816-21.
  5. Karvelis T, et al. 2013. Biochem Soc Trans. 41(6): 1401-6.
  6. Wang H, et al. 2013. Cell. 153(4): 910-8.
  7. Kaminski R, et al. 2016. Gene Ther. 23(8-9): 690-5.
  8. Kaminski R, et al. 2016. Scientific Reports. 6:22555.
  9. Zhan T, et al. 2018. Seminars Cancer Biology. S1044-579X(17)30274-2.
Contributed by Christopher Dougher, PhD.
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