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CRISPR technology: from nowhere to everywhere

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The first thing to say about a technique that has been hailed as ground-breaking, particularly in the fields of health and agriculture, and is attracting both high praise and visceral opposition, is that it was not invented but discovered and adapted.

All living organisms have the problem of staying alive. In the late 1980s a group of scientists discovered that the DNA in bacteria contains sequences in a specific pattern: short, repeating, palindromic DNA sequences (a palindrome is a word, phrase, number, or other sequence of characters which reads the same backward or forward) separated by a short, non-repeating, spacer DNA sequences. These they called “Clustered Regularly Interspaced Short Palindromic Repeats”, or CRISPR for short. More work revealed that CRISPR, together with a set of proteins called Cas (for CRISPR-associated) and specialised RNA molecules, are part of a basic bacterial immune system that recognises specific sequences in the DNA of attacking viruses and cuts them in two to halt infections.

In 2012 it was announced that the CRISPR-Cas system had been adapted to identify, cut and even replace any sequence in most organisms: a gene-editing tool with an unprecedented level of power and precision. And the technology seemed simple and affordable enough to have encouraged even amateur, and possibly ill-intentioned, DIY biologists to try their hand on it (“Biohackers gear up for genome editing”).

Figure 1: CRISPI-Cas publications up to July 2015.
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Research on applications of the technology and on how to improve it started shortly afterwards. Initially new scientific publications on the topic trickled in (fig. 1). But suddenly CRISPR is everywhere, with 1288 peer-reviewed articles published in 2015 (Pubmed).

CRISPR is likely here to stay. While powerful new technologies come with many promises and potential, they also bring concerns. The key point of dissent with respect to CRISPR is whether research in human germ cells (the cells that give rise to the sperm and eggs) and embryos should be allowed to proceed. This has even divided the scientific community. There are powerful arguments and ethical considerations on both sides of the argument. Should we be taking the risk of making mistakes with humans? Can CRISPR make changes to the DNA in places it is not designed to do so? And if yes, with what frequency? Yet at the same time we need to consider whether we can justify not using a tool that could cure critical diseases and bring many other benefits to humans.

A debate on the role of CRISPR in and by humans, within the wider context on how the use of new technologies should be assessed and regulated, is critical.

How does CRISPR work?

One of the first clues about the role of CRISPR in bacterial immunity was the fact that the DNA found in the spacer sequence was the same as that of some viruses. This suggested that bacteria were incorporating viral DNA pieces in their chromosomes to ‘remember’ attacking viruses and targeting them for destruction, as a form of ‘molecular memory’. This turned out to be the case.

(Box 1 lists the background knowledge needed to understand CRISPR technology.)

Box 1: 

1: DNA is a long, chain of two strands formed by individual units called nucleotides: adenine, thymine, cytosine and guanidine (A, T, C and G). The nucleotides of one strand bind the nucleotides of the other strand in a specific pattern: A to T and C to G. The sequence of the two strands is therefore complementary, not identical. Because double-stranded DNA is much more stable than single stranded DNA, single stranded DNA molecules in the cell bind (or pair up with) their complementary strands.

2: Genes are discrete units in the DNA strand and most genes contain the information to make a specific protein. When genes are ‘turned on’ (or ‘expressed’), one of the strands is copied to make another molecule called RNA. RNA, like DNA, is also made up of nucleotides (not quite the same ones), is ‘selectively sticky’, and can bind to DNA.

3: The second stage of gene expression involves ‘translating’ the information in the RNA molecule to make a protein (although some RNA molecules are never translated).

4: This sequence-specific ‘stickiness’ of DNA and RNA molecules is a key feature, and the basis of many important laboratory techniques.   

This is how it works:

1. A set of Cas proteins (Cas1/Cas2) is tasked to recognise viral DNA pieces and incorporate them as spacers in the CRISPR DNA.

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2. The spacers are then ‘read’ to make a short RNA molecule (called crRNA) which is incorporated into another Cas protein, Cas 9.

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3. The Cas9 complex is guided by the crRNA to the target viral DNA pieces, cutting them to stop infection.

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CRISPR as a laboratory tool
The way the CRISPR system was modified to become a gene-editing tool was by making in vitro CRISPR systems where the chosen gene target is incorporated as a spacer. These are introduced, along with the Cas9 gene, into the organism of choice (plant, animal, or even human cells) by genetic modification (GM) to edit the gene of interest. Depending on the specific CRISPR system used, the outcome will be small insertions, deletions or inversions that modify or stop gene expression.

In addition, gene sequences can be precisely changed by introducing a template DNA sequence because these are copied by the cell’s repair kit and incorporated in the DNA when it ‘fixes” the DNA breaks made by Cas9 in a process called ‘homology-directed recombination’.

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A CRISPR timeline

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How are plants and animals developed by CRISPR regulated?

Although CRISPR requires an initial GM step, the Cas9 protein and guide RNA are needed only once. So, if used to improve crops or livestock, the inserted CRISPR-Cas9 transgene (a transgene is the foreign DNA inserted in an organism by GM) can be removed in subsequent generations by selecting individuals with the desired changes but lacking the CRISPR-Cas9 gene. What is more, a DNA-free CRISPR system has also been developed: the mix of proteins needed for gene edited are added as reagents and introduced into plant cells using solvents. So the final gene-edited product may differ from the original unimproved plant or animal only by a few nucleotides (undistinguishable from the effects of natural sequence differences due to evolution) and also lack any foreign DNA. Therefore plants and livestock produced by CRISPR are not GM. Or are they? This is a currently a hotly debated topic.

Deliberations on how products developed by the so-called New Plant Breeding Technologies (NPBTs), which include CRISPR, should be regulated in the EU have been ongoing for several years. In 2007 the European Commission appointed an expert working panel to review eight of the latest toolset of plant breeding technologies to guide their mode of regulation. However, by the beginning of 2015 no decision had been reached. In February 2015 the German Federal Office of Consumer Protection and Food Safety (BVL) announced that an oilseed variety developed by a commercial company using a gene-editing technique was not to be classified as a GM organism and they have more recently confirmed their stance in this matter. The Swedish Board of Agriculture has also stated that some plants in which the genome has been edited using the CRISPR-Cas9 technology do not fall under the European GMO definition and the UK Advisory Committee on Releases to the Environment (ACRE’s) has reached the same conclusion. However, the European Commission has still to take a position on these technologies and the result is that these technologies are stuck in a legal limbo.

Lack of transparency on the regulatory status of the newest set of plant breeding tools and the overregulation of crops developed by them would severely limit their use and potential, argues the scientific community. Most affected will be applications in the public sector and research in so-called ‘orphan crops’ which are important for developing countries in terms of food security and adequate nutrition but not or little traded in the international markets. Investment for research in the latter tends to be much lower than for internationally traded crops. And there are also some practical considerations: the changes caused by NPBTs are at times indistinguishable from naturally occurring genetic variation, so there would be no way of distinguishing the two experimentally.

The Nuffield Council on Bioethics announced an open call for evidence closing on 1 February 2016 on the use of genome editing, with separate sections for plants, animals, microorganisms and human medical research. The main aim of the call is “to identify and define ethical questions raised by recent advances in biological and medical research in order to respond to, and to anticipate, public concern.”

The United States has also exempted a number of crops developed by the newest set of breeding technologies and existing regulations for crops developed by biotechnology tools are also under review. The current regulatory trigger in the USA is the use of a bacterium (Agrobacterium) to insert DNA into crops by GM, even if the final products no longer carry any foreign DNA.

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