The Crispr-Cas system is a genome editing technique that allows to alter the genetic code of any given organism. The method, which is derived from an inherent adaptive immune system in bacteria and archaea, was first published in 2012 and has since taken the scientific community by storm. Owing to its simplicity and efficiency it quickly developed into a mainstream method and was adapted by labs all over the globe.

In many ways it has since entirely changed the way genome engineering is done. The days when it took a tenacious Post-Doc years to develop a genetically engineered mouse strain are definitely over.

As all the buzz and excitement in the scientific community swapped over into the public and it became clearer in how many ways CRISPR could be used, many people also began to worry about potential risks. Over the last few years genetically modified organisms have been introduced into nature and there has been talk of editing the human germline.

Below you can find my summary of what CRISPR-Cas is all about, a description of how it works, applications to date and possible risks. I will continue to add to this factsheet over the years to make sure that it is kept up to date – if you have any questions or suggestions on what could be added, feel free to list them in the comments.

What is it and where did it come from?

• CRISPRs are Clustered Regularly Interspaced Short Palindromic Repeats.
• They are short DNA sequences, each about 30 bases long, read similarly backwards and forwards and repeat every 35 bases or so. ¹
• They are widely distributed among bacteria and archaea, which use them to identify and destroy invading viruses. ¹
• The DNA in the spaces between the palindromic repeats (spacers) matches, for example, viral sequences. CRISPR-associated enzymes (Cas proteins) add these sequences to the bacterial genome after infection. ¹
• RNA made from these spacers can bind to invading DNA or RNA from viruses or other pathogens. Cas detects these bound fragments and cuts the DNA to inactivate the gene. ¹
• So in a way the spacers are a library of foreign DNA signatures from invading pathogens and other material. A CRISPR array typically contains fewer than 50 units of these spacer-repeat sequences.
• Scientists assume that the CRISPRs have roles besides host defence as well, such as genetic regulation of group behaviour and virulence, DNA repair and genome evolution. ²
• Compared to other bacterial defence mechanisms, such as restriction enzymes which can also cut DNA at a certain sequence, CRISPRs are much more adaptive and highly dynamic.

How does the CRISPR technology work?

• The most commonly used CRISPR-Cas9 type II system consists of the enzyme Cas9, which can cut DNA double-strands at a defined position, and a piece of guide RNA (gRNA) that binds to this position in the DNA and tells Cas9 where it has to go.
• The gRNA bases are complementary to those in the gene region that is targeted. In theory, this region should be unique in the genome, preventing any further binding elsewhere (off-target).
• Once the gRNA is bound to the genome, Cas9 attaches itself to the gRNA and the DNA underneath and cuts one or both strands, depending on its function.
• The DNA damage is then noticed by the cell, which recruits repair enzymes to seal it up again. If a DNA template is provided, a new genomic region can now be inserted during the repair. This way researchers can add new genetic material or correct mutations. It is also possible to simply cut and deactivate a gene entirely.
• A simplified version of the CRISPR-Cas mechanism of action can be found on the website of the Royal Society of Biology.
• The full scientific explanation can be found here [4].

What can it do?

• The Cas9 enzyme can add mutations, it can replace or add specific gene sequences or delete them. And It can rearrange the genomic code (called inversions or translocations).
• By fusing a colour-tag to a certain gene sequence this region can be labelled and tracked under a microscope.
• By fusing Cas9 to a so-called activator domain it can also actively control the expression of genes, for example also through introducing epigenetic changes.
• For a more scientific explanation of all it’s features have a look at this Review. [3]

What are potential and current applications?

• Humans: In theory any disease caused by a genetic mutation should be tractable with CRISPR, including cancer or devastating congenital disorders such as sickle-cell anaemia or cystic fibrosis. Inherited diseases could be possibly avoided by genetic modifications to embryos or sperm and egg cells from parents – so-called germline editing. Due to the enormous ethical and legal implications, the 2017 consensus of the US National Academies of Science, Engineering, and Medicine concludes that germline editing should not be performed on embryos intended for establishing a pregnancy. Editing human embryos for research purposes is allowed.
• Plants: Compared to conventional GM organisms, in which scientists cannot control where they add the gene, CRISPR is much more accurate and versatile. By knocking out several genes at once using CRISPR or TALENS, a separate technique, scientists have produced rice that is inherently resistant to bacterial leaf streak and blight, two severe diseases. Mushrooms have been modified to resist browning and there are a number of biotech companies currently working on similar procedures in other plant species.
• Insects: Scientists have engineered the Anopheles mosquito, which is known to spread malaria, to pass on genes in female offspring that can cause infertility. Using so-called gene drives – inserting DNA that encodes CRISPR-Cas9 with a mutated gene and can be copied from one chromosome to another – they get around evolutionary mechanisms that would prevent infertility mutations from spreading. A different lab has also developed a species that can spread a malaria-inactivating antibody through a population. However, more recently it emerged that mosquitoes develop resistances to the gene drive. The main reasons for that might be the extensive natural genetic variation among mosquitoes and the fact that CRISPR will only target a few fertility genes at once.
• Mammals: Among recent studies we have pigs that are immune to the porcine reproductive and respiratory syndrome virus. Micropigs that can be kept as pets. Chicken that produce eggs with reduced allergen content. What is up next in the CRISPR zoo?

In which species has it been used so far?

• CRISPR has been used in: human embryos, human cells, rhesus monkey embryos, mice, pigs, chicks, rats, rabbits, frogs, zebrafish, fruit flies, roundworms, silk worms, rice, wheat, sorgum, tobacco, thale cress, barley, yeast and bacteria. [3] Please note that due to the vast increase in literature this list is not complete.

What are the risks and implications

• Human germline editing: As any change made in germline cells will be passed on from generation to generation it has important ethical implications and is currently illegal.
• Ecological risks: If gene drives indeed succeed in wiping out entire insect species the consequences for the ecosystem in affected regions could be dire and unforeseeable. The same of course also applies for any changes made to plant populations or, indeed, animals.
• Off-target effects: mutations or other changes might be induced in regions of the genomic code that were not targeted. For example, sequences might be cut that are similar to the targeted sequence and could lead to defects in essential genes which might cause disease. Off-target effects can be reduced by using more complex cocktails of CRISPR components, including additional RNA sequences, different types of gRNA or Cas enzymes with specific functions. CRISPR technology also needs to be optimised for each cell type.
• Home use: As CRISPR technology is easy to use and relatively cheap, many experts are warning of a risk to society due to uncontrolled use in “home-based” labs using genome editing kits.

Want the scientific details? Here are the references:

1. Nature. 2012 Feb 15;482(7385):331-8. doi: 10.1038/nature10886. RNA-guided genetic silencing systems in bacteria and archaea. Wiedenheft B, Sternberg SH, Doudna JA.
2. Nat Rev Microbiol. 2014 May;12(5):317-26. doi: 10.1038/nrmicro3241. Epub 2014 Apr 7. Review. CRISPR-Cas systems: beyond adaptive immunity. Westra ER, Buckling A, Fineran PC.

3. Nature Biotechnology 32, 347–355 (2014) doi:10.1038/nbt.2842. CRISPR-Cas systems for editing, regulating and targeting genomes. Jeffry D Sander & J Keith Joung.

4.  Nature Biotechnology34,933–941(2016)doi:10.1038/nbt.3659. Applications of CRISPR technologies in research and beyond. Rodolphe Barrangou & Jennifer A Doudna

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