RESEARCH IN PRACTICE
What is CRISPR/Cas9?
Melody Redman,1 Andrew King,2 Caroline Watson,3 David King4
1
HYMS Centre for Education INTRODUCTION currently only been validated in preclin-
Development (CED), Hull York
Clustered regularly interspaced palin- ical models, but there is hope it can soon
Medical School, University of
York, York, UK dromic repeats (CRISPR)/Cas9 is a be translated to clinical practice.
2
Molecular Haematology Unit, gene-editing technology causing a major Schwank et al used CRISPR/Cas9 to
MRC Weatherall Institute of upheaval in biomedical research. It makes investigate the treatment of CF. Using
Molecular Medicine, University
of Oxford, John Radcliffe
it possible to correct errors in the genome adult intestinal stem cells obtained from
Hospital, Oxford, UK and turn on or off genes in cells and organ- two patients with CF, they successfully
3
Department of Haematology, isms quickly, cheaply and with relative corrected the most common mutation
Oxford University NHS ease. It has a number of laboratory applica- causing CF in intestinal organoids. They
Foundation Trust, Churchill
Hospital, Oxford, UK
tions including rapid generation of cellular demonstrated that once the mutation had
4
Academic Unit of Child Health, and animal models, functional genomic been corrected, the function of the CF
Sheffield Children’s Hospital, screens and live imaging of the cellular transmembrane conductor receptor
Sheffield, UK genome.1 It has already been demonstrated (CFTR) was restored.4
Correspondence to that it can be used to repair defective DNA Another disease in which CRISPR/Cas9
Dr David King, Academic Unit of in mice curing them of genetic disorders,2 has been investigated is DMD. Tabebordbar
Child Health, Sheffield Children’s and it has been reported that human et al recently used adeno-associated virus
Hospital, Western Bank,
embryos can be similarly modified.3 Other (AAV) delivery of CRISPR/Cas9 endonu-
Sheffield S10 2TH, UK;
[Link]@[Link] potential clinical applications include gene cleases to recover dystrophin expression in
therapy, treating infectious diseases such as a mouse model of DMD, by deletion of the
Received 5 January 2016 HIV and engineering autologous patient exon containing the original mutation. This
Revised 18 February 2016
Accepted 19 February 2016
material to treat cancer and other diseases. produces a truncated, but still functional
Published Online First In this review we will give an overview of protein. Treated mice were shown to par-
8 April 2016 CRISPR/Cas9 with an emphasis on how it tially recover muscle functional deficien-
may impact on the specialty of paediatrics. cies.5 Significantly, it was demonstrated that
Although it is likely to have a significant the dystrophin gene was edited in muscle
effect on paediatrics through its impact in stem cells which replenish mature muscle
the laboratory, here we will concentrate on tissue. This is important to ensure any
its potential clinical applications. We will therapeutic effects of CRISPR/Cas9 do not
also describe some of the difficulties and fade over time. Two similar studies have
ethical controversies associated with this described using the CRISPR/Cas9 system in
novel technology. vivo to increase expression of the dys-
trophin gene and improve muscle function
OVERVIEW OF CRISPR/CAS9 in mouse models of DMD.6 7 Other studies
CRISPR/Cas9 is a gene-editing technology have used CRISPR/Cas9 to target duplica-
which involves two essential components: tion of exons in the human dystrophin gene
a guide RNA to match a desired target in vitro and have shown that this approach
gene, and Cas9 (CRISPR-associated can lead to production of full-length dys-
protein 9)—an endonuclease which causes trophin in the myotubules of an individual
a double-stranded DNA break, allowing with DMD.8
modifications to the genome (see CRISPR/Cas9 could also be used to
figure 1). treat haemoglobinopathies. Canver et al9
recently showed BCL11A enhancer dis-
POTENTIAL CLINICAL USES ruption by CRISPR/Cas9 could induce
Correction of genetic disorders fetal haemoglobin in both mice and
One of the most exciting applications of primary human erythroblast cells. In the
CRISPR/Cas9 is its potential use to treat future such an approach could allow fetal
genetic disorders caused by single gene haemoglobin to be expressed in patients
To cite: Redman M, King A,
mutations. Examples of such diseases with abnormal adult haemoglobin. This
Watson C, et al. Arch Dis include cystic fibrosis (CF), Duchenne’s would represent a novel therapeutic strat-
Child Educ Pract Ed muscular dystrophy (DMD) and haemo- egy in patients with diseases such as
2016;101:213–215. globinopathies. The approach so far has sickle cell disease or thalassaemias.
Redman M, et al. Arch Dis Child Educ Pract Ed 2016;101:213–215. doi:10.1136/archdischild-2016-310459 213
Research in practice
and stem/progenitor cells which can then be reintro-
duced into patients to treat disease. This approach
may overcome some of the issues associated with how
to efficiently deliver gene editing to the right cells.
T-cell genome engineering has already shown
success in treating haematological malignancies and
has the potential to treat solid cancers, primary
immune deficiencies and autoimmune diseases.
Genetic manipulation of T-cells has previously been
inefficient. However, Schumann et al recently
reported a more effective approach in human CD4+
Figure 1 The CRISPR/Cas9 system.1 Clustered regularly
T-cells based on the CRISPR/Cas9 system. Their tech-
interspaced palindromic repeats (CRISPR) refers to sequences in nique allowed experimental and therapeutic knock-
the bacterial genome. They afford protection against invading out and knock-in editing of the genome in primary
viruses, when combined with a series of CRISPR-associated human T-cells. They demonstrated T-cells could be
(Cas) proteins. Cas9, one of the associated proteins, is an manipulated to prevent expression of the protein
endonuclease that cuts both strands of DNA. Cas9 is directed to PD-1, which other work has shown may allow T-cells
its target by a section of RNA. This can be synthesised as a
to target solid cancers.11
single strand called a synthetic single guide RNA (sgRNA); the
section of RNA which binds to the genomic DNA is 18–20
There is also interest in using CRISPR/
nucleotides. In order to cut, a specific sequence of DNA of Cas9-mediated genome editing in pluripotent stem
between 2 and 5 nucleotides (the exact sequence depends cells or primary somatic stem cells to treat disease. For
upon the bacteria which produces the Cas9) must lie at the 3’ example Xie et al12 showed the mutation causing
end of the guide RNA: this is called the protospacer adjacent β-thalassaemia could be corrected in human induced
motif (PAM). Repair after the DNA cut may occur via two pluripotent stem cells ex vivo. They suggest that in
pathways: non-homologous end joining, typically leading to a
the future such an approach could provide a source of
random insertion/deletion of DNA, or homology directed repair
where a homologous piece of DNA is used as a repair template.
cells for bone marrow transplantation to treat
It is the latter which allows precise genome editing: the β-thalassaemia and other similar monogenic diseases.
homologous section of DNA with the required sequence change
may be delivered with the Cas9 nuclease and sgRNA, LIMITATIONS
theoretically allowing changes as precise as a single base-pair. A number of challenges remain before the potential of
CRISPR/Cas9 can be translated to effective treatments
Knock-in of a fully functional β-globin gene is much at the bedside. A particular issue is how to deliver
more challenging, which is the reason for this some- gene editing to the right cells, especially if the treat-
what unusual approach. ment is to be delivered in vivo. To safely deliver
Cas9-nuclease encoding genes and guide RNAs in
Treatment of HIV vivo without any associated toxicity, a suitable vector
Another potential clinical application of CRISPR/Cas9 is needed. AAV has previously been a favoured option
is to treat infectious diseases, such as HIV. Although for gene delivery.1 However, this delivery system may
antiretroviral therapy provides an effective treatment be too small to allow efficient transduction of the
for HIV, no cure currently exists due to permanent Cas9 gene.1 A smaller Cas9 gene could be used, but
integration of the virus into the host genome. Hu et al this has additional implications on efficacy.1 A number
showed the CRISPR/Cas9 system could be used to of other non-viral delivery systems are under investi-
target HIV-1 genome activity. This inactivated HIV gation and this process requires further optimisation.
gene expression and replication in a variety of cells Another significant concern is the possibility of off-
which can be latently infected with HIV, without any target effects on parts of the genome separate from
toxic effects. Furthermore, cells could also be immu- the region being targeted. Unintentional edits of the
nised against HIV-1 infection. This is a potential thera- genome could have profound long-term complications
peutic advance in overcoming the current problem of for patients, including malignancy. The concentration
how to eliminate HIV from infected individuals. After of the Cas9 nuclease enzyme and the length of time
further refinement, the authors suggest their findings Cas9 is expressed are both important when limiting
may enable gene therapies or transplantation of genet- off-target activity.1 Although recent modifications in
ically altered bone marrow stem cells or inducible the nuclease have increased specificity, further work is
pluripotent stem cells to eradicate HIV infection.10 required to minimise off-target effects and to establish
the long-term safety of any treatment.
Engineering somatic cells ex vivo to treat malignancy The therapeutic applications of CRISPR/Cas9 con-
or other diseases sidered in this article have predominantly been direc-
There has been increasing interest in the possibility of ted at somatic cells. A particularly controversial issue
using CRISPR/Cas9 to modify patient-derived T-cells surrounding CRISPR/Cas9 is that of gene editing in
214 Redman M, et al. Arch Dis Child Educ Pract Ed 2016;101:213–215. doi:10.1136/archdischild-2016-310459
Research in practice
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Redman M, et al. Arch Dis Child Educ Pract Ed 2016;101:213–215. doi:10.1136/archdischild-2016-310459 215
