CRISPR-cas System: Overview
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)-cas is a recently described gene-editing synthetic biology tool which takes advantage of the bacterial immune system. It is cheap, simple, easy, faster, more precise, quickly evolving, and more adaptable as compared to other methods used for gene manipulation e.g. TALENS, Zinc-Finger Nucleases, MAGE (Multiplex Automated Genome Engineering) etc.
How CRISPR-cas9 works:
CRISPRs are repeating sequences found in the bacterial genetic code interspersed with ‘spacers’ – unique stretches of DNA that the bacteria acquire from the invading viruses. On a repeat encounter with the viruses, the bacteria can recognize this DNA sequence and transcribe matching RNA (crRNAs) using the archived ‘spacer’ material that guide the effector nucleases to locate and cleave the nucleic acids complementary to the spacer.
There are 3 major types of CRISPR systems-I-III. The type II CRISPR-cas9 system derived from Streptococcus pyogenes is best characterized and is most widely used for gene editing. The system consists of 2 components:
- Cas9, a non-specific CRISPR-associated endonuclease and
- gRNA, a short synthetic guide RNA composed of:
- A ‘scaffold’ sequence necessary for Cas9 binding and
- ~20 nucleotide ‘spacer’ sequence targeting the genomic sequence to be modified. Oligo pools of single-guide RNA, sgRNA, can be generated by PCR amplification and delivered as such or cloned into plasmids or viral vectors, or used with nanoparticles.
- gRNA joins up with the DNA-cutting cas-9 enzyme
- RNA aligns with the target DNA and cas9 cuts the double helix
- DNA repair and precise sequence changes are triggered.
Upon the cleavage by cas9, the target DNA is repaired either by non-homologous end-joining (NHEJ) or by homology directed repair (HDR). If not repaired, the double-stranded break (DSB) re-ligates through the NHEJ process leading to random insertion/deletion mutations. In contrast, HDR causes targeted gene deletions, insertions, mutations or corrections by homologous recombination using the donor DNA template.
The CRISPR-cas tools themselves present little or no risk to the research staff or the public. However, the outcomes of gene editing experiments have caused ethical concerns including:
- Human integrity: Germ-line alterations are debated for their intentions and potential outcomes e.g. adverse effects may not be noticed until birth or later in life, lack of consent from the subsequent generations, making ‘designer babies’ etc.
- Ecosystem integrity: CRISPR Gene Drive technology can spread certain traits through a population of sexually reproducing organisms. In experiments with Drosophila, mosquitoes, and yeast, gene drive systems spread changes through wild populations, converting the heterozygotes into homozygotes in each generation. It is important to consider unknown risks to both target and non-target species as well as the entire ecosystem and consider issues such as dual-use potential, selection of release sites, governance, adequate oversight, and international impacts.
- Biosafety: Accidental exposures of laboratory workers to CRISPR-cas material could result in the insertional activation of oncogenes, a risk of inactivation of tumor suppresser genes etc. The use of replication competent viruses to deliver CRISPR-cas to host cells or organisms adds to the risk associated with gene editing experiments. The escape of uncharacterized gene-edited organisms from containment is also a concern.
- Biosecurity: There is a heightened possibility of creating potentially harmful biological agents e.g. a dual use concerns such as introducing antibiotic resistance, increasing disease potential or virulence in pathogens, toxin production in crops etc. Gene editing research may be conducted by persons or organizations with bad intent or by countries that may have less stringent ethical and/or regulatory standards.
Many modified plants are non-regulated. On July 2, 2015, the White House issued a memorandum directing the EPA, FDA, and USDA to “develop a log-term strategy so that the agencies are prepared for the future biotechnology products”.
2012: Mol. Biologist Jennifer Doudna, UC, Berkeley & Microbiologist Emmanuelle Charpentier, Umea University, Sweden and the Max Planck Institute for Infection Biology, Berlin published a Science paper demonstrating that the Cas9 enzyme can be directed to cut specific sites in isolated DNA.
2013: Feng Zhang, Broad Institute of MIT & Harvard, Cambridge, MA published a Science paper demonstrating the CRISPR-cas9 application in mammalian cells.
Between 2008 and 2014, the number of publications on use of CRISPR-cas system increased from 0-~1500, the number of patents from 0-160 and the funding for research rose from $ 0-90 million.
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11. From presentations at the Harvard-Yale IBC Symposium , 3 May 2016