This year marks the tenth anniversary of the seminal paper by Charpentier and Doudna in Science (1) on what can be considered one of the most significant breakthroughs in the field of genetic engineering. In the ensuing years, CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats, has taken the world by storm. In this article, we provide an overview of this outstanding technology – from basic biology to the future almost science fictionesque applications.
What is CRISPR?
Let us start with a birds-eye view. CRISPR, or more appropriately, the CRISPR-Cas9 system (CAS: CRISPR associated protein) allows us to cut DNA at specific sites in the genome. It also allows us to paste DNA sequences of our choice into specific sites in the genome. Although we have had the ability to cut DNA in specific sites before, there are many significant advantages that the CRISPR-CAS9 has, making it the most powerful gene-editing system to date. Firstly, it allows us to change DNA sequences in situ – which means we can make specific changes directly in the genome. Moreover, it is easy to design and implement, and most significantly, it has been shown to be effective in several plant and animal species. Put together, these properties make this system ominously powerful.
A bit more into the specifics.
The acronym CRISPR expands to Clustered Regularly Interspaced Short Palindromic Repeats, which
does little in the way of explanation. Indeed, one would struggle to connect the name with its mode
of operation or its astounding array of applications. The acronym, CRISPR, actually refers to parts of
the bacterial genome – stretches of DNA that exhibit these characteristic patterns of repeating
palindromes separated by random nucleotide sequences that till recently no one knew the function
of. Palindromes are sequences of DNA that read the same whether they are read front to back or back
to front – like the word Malayalam or tenet. An example of a palindromic DNA sequence is GAATTC (Figure 1). Palindromic “restriction” sites and
associated “restriction endonucleases" were discovered way back in 1970 (2) and the CRISPR locus was first elucidated in the late 1980s (3). Over the
years, researchers discovered that this system is part of an elaborate immune mechanism in-built into bacteria and archaea.
Viral DNA is injected into bacteria when they are infected by viruses. Bits of viral DNA are integrated
into the bacterial genome in between palindromic sites. This stretch of the bacterial genome also
encodes for the CRISPR-associated protein. When bacteria are invaded by the virus again, this system
kicks in. The stretch of DNA is transcribed into what is known as crRNA and the Cas protein is expressed
An example of a palindromic sequence – together they form a nucleoprotein complex that recognizes specific sequences of the invading viral
DNA that are complementary to the ones that were stored in the CRISPR system earlier. Once the RNA hybridizes with the viral DNA at these specific sites, the Cas swings into action and
creates double-stranded breaks (DSB) in the viral DNA. These DSBs can be filled via the
process of Non-Homologous End Joining (NHEJ), in which the cell apparatus fills in the
gap formed by adding random nucleotides causing so-called Indel mutations that disrupt
gene expression. This process can also cause the DNA to be processed for degradation.
Alternatively, the break can be filled in with a donated DNA template via Homology Directed Repair
(HDR), which allows us to insert specific DNA sequences of interest into the genome. In summary, the
CRISPR-Cas system provides an RNA-guided antiviral immunity, and the immunity is first gained upon
exposure to invading genetic elements. Therefore, CRISPR-Cas codes for an adaptive immune system.
The real revolution was kickstarted by the discovery that this system of RNA-guided DNA cuts could
be adapted and used in a eukaryotic cell in a programmable fashion to create double-stranded breaks
at specific sites of interest. Based on the newer Type II systems, which used another stretch of guide
RNA called Trans-activating RNA (tracrRNA) in addition to the crRNA, composite RNA guides were
created that could direct a single Cas9 protein to desired sites in the DNA (1). Furthermore, it was
quickly discovered that this system worked pretty across many plant and animal species. And most
significantly, it was demonstrated that this system could be used to replace stretches of DNA in situ
when combined with an externally provided DNA template. Although there have been other methods
to achieve this, the ease with which CRISPR-Cas allows us to create gene edits makes it one of the
most potent tools to date. Evolution – a process that has happened over millennia in a relatively slow
manner, reacting to mainly local evolutionary pressures, now can be directed and achieved almost
overnight. This ability gives us humans, arguably the most fickle-minded species on the planet, the
power to play God.
The Good, bad and ugly
There is no doubt that this technology has astounding medical potential. CRISPR-Cas is one of the
latest entrants to the field of gene therapy – the use of DNA/ RNA as therapeutic molecules. Gene
CRISPR-Cas9: Using the sgRNA, the Cas9 protein
finds corresponding targets in the DNA and
creates double-stranded breaks (DSBs) that can
be filled non-specifically (NHEJ) or by inserting a
piece of DNA template (HDR).
therapy can involve adding a missing gene into the cell. The added gene will produce the functional
protein required to overcome the defect. Alternatively, we could interfere with the transcription
process using approaches such as anti-sense oligonucleotides or RNAi to prevent a particular gene
from expressing. CRISPR takes it one step further by allowing us to change the gene sequence directly
in the genome. It belongs to the category of “gene editing systems”, which include older modalities
such as Zinc-Finger-Nucleases (ZFN) and Transcription Activator–Like Effector Nucleases (TALENs). Not
only does this process allow us to turn on or off specific genes in the genome, but it also allows us to
correct gene defects (such as mutations, insertions, and deletions) so that the correct protein is
expressed. Correcting gene defects obviously has tremendous medical value to help millions of people
suffering from inherited gene disorders.
Single-gene disorders such as Muscular Dystrophy, Spinal Muscular Atrophy, Cystic Fibrosis, and βthalassemia are attractive targets of gene therapy because theoretically, we could effect a cure by correcting a single gene defect (4,5). However, we could even use CRISPR to develop treatments for
multi-gene diseases. For example, genetically engineered T cells are all the rage in cancer therapy and were one of the first applications to be demonstrated experimentally using CRISPR (6). Chimeric Antigen Receptor-T cells (CAR-T cells) – patients’ own CD8+ T cells that are obtained and genetically modified to introduce an antigenic target on their cell surface – have shown great promise in treating refractory B-cell lymphoma. In fact, the future of biomedical technology will heavily favour the use of genetically engineered cells that could help us treat diverse diseases such as AIDS, cancer, and autoimmune disease (7–9).
Another major application of CRISPR is in disease modelling. Many complex diseases such as
Amyotrophic Lateral Sclerosis still do not have a good animal model to study the disease. CRISPR has
made it easier to create transgenic animals that can be used to understand the disease. Since it has
been shown that CRISPR can be used to edit multiple genes simultaneously (10,11), the method can
be used to study the interaction of different genes on disease outcomes. For example, Maresch et al
have demonstrated the power of multiplexed gene editing achieved through CRISPR to create a mice
model of pancreatic cancer (10). Additionally, the combination of CRISPR with Induced Pluripotent
Stem Cells provides a powerful platform for disease modelling. For example, researchers have used
human-IPSC-derived kidney cells to generate “spheroids”, 3D cell clusters that mimic organs more
closely than 2D cultures. These spheroids were then gene-edited using CRISPR-Cas9 to generate
knockouts of PKD1 and PKD2 genes, which resulted in the formation of cysts. This system can now be
used in further studies of kidney diseases (12).
Apart from biomedical applications, CRISPR has obvious applications in plant biotechnology, where it
has been used to create crops with increased yield, disease resistance, improved quality, and herbicide
resistance (13). Also, this technology will have a significant impact on animal husbandry – helping
create an improved quality of livestock with better resistance to disease, improved fertility, and overall
health (14).
Gene therapies usually work with somatic cells and in post-natal tissues. However, gene editing does
not have to be limited to somatic tissues. When CRISPR-like technologies are used to create germline
modifications, it opens up a Pandora’s Box of ethical ramifications. And the most problematic issue is
that gene edits can be designed to be inheritable. In fact, these gene edits could exploit the naturally
occurring phenomenon of gene drives – genetic elements that promote their own transmission to the
next generation (15,16). Some researchers are advocating the use of gene drives to solve major
environmental problems such as malaria and rodent infestation (17). Synthetic gene drive systems can
be introduced into key species, to modify or eliminate the targeted populations. While on the surface
this appears to be a great way to tackle a severe problem, it comes with huge ethical responsibilities.
The fact that it could potentially wipe off a particular species, even if that is an invasive and
problematic species has huge ramifications. How that will affect the ecosystem is the big question.
Nevertheless, these experiments have already started in a couple of places in the world.
What lies ahead
The future of genetic engineering is full of exciting and sometimes scary possibilities. Changing the
genetic code of organisms has never been easier. There are, of course, some technical challenges that
have to be overcome. One of the most relevant is the fact that CRISPR-Cas can cause unwanted gene
edits in non-targeted regions of the gene. Although much better than older systems, it is still a cause
for concern as it opens up the possibility of tumorigenesis (18). The other major challenge is common
to all gene therapy approaches – that of delivery. Getting the CRISPR-Cas into enough cells to bring
about an effective cure is still a challenge (19). Most programs are still betting on virus-based
approaches with Adeno-Associated Virus (AAV) as the favourite (20). Although AAV provides several
advantages, it has limited capacity and can induce immunogenicity in humans. Non-viral approaches
such as polymers, lipids, and electroporation have been tried and are fairly successful in ex vivo gene
editing – for example, electroporation has been used for transfecting T cells to create CAR-T (6).
However, these approaches are not efficient enough or not suitable for direct in vivo applications.
Newer versions of Cas have been discovered to further fine-tune the targets. An example is Cas13,
which edits RNA instead of DNA - a feature that allows temporary changes rather than permanent
genetic changes that would occur in genomic DNA (21).
The future, however, may well be in controlling our genetic destiny. Designer babies have been in
popular media for a while (22,23). Although germline genome editing has been officially banned,
CRISPR has already been used to create genetically edited babies – in this instance, creating babies
that were supposedly resistant to HIV infection (24). The perceived ease of using CRISPR has brought
it to popular culture with many advocating democratizing the technology and that it be freely available
to everyone for use. Surely, such powerful technology in the hands of a few rich and power-hungry
people would be a recipe for a dystopian world. At the same time, widespread unregulated use of
such technologies could be equally problematic.
Advances in technology invariably bring with them the power and responsibility of choice. We humans
who have inherited the earth have the choice to utilize the power to preserve and flourish or to
destroy and extinguish. It is, of course, a fervent hope that the human race will have the intelligence
to choose what’s best and perhaps, more importantly, know what’s best.
About the author
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Dr. Gururaj A Rao, Managing Director, International Stemcell Services Ltd,Bengaluru
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LSW LifeScienceWorld