Demystifying the Lab
ASH Clinical News takes a look at the complex scientific techniques that
hematologists/oncologists hear about every day, with practical information
for the practicing clinician.
DEMYSTIFYING
Gene Editing With CRISPR
Gene editing has received a great deal of media
attention in recently due to its potential for
advancing science and treating disease. In a
short period of time, scientists have developed
several powerful tools that are capable of intro-
ducing extremely precise genomic alterations.
In particular, clustered regularly interspaced
short palindromic repeats (CRISPR) and
CRISPR-associated protein 9 (Cas9), which was
named Science’s 2015 Breakthrough of the Year
and for which the developers received several
prestigious awards, has been widely adopted
by scientists due to its relative affordability, ef-
ficiency, and ease of use. 1
Gene therapy is predicted eventually to
benefit millions of patients by enabling shorter
treatment regimens with longer-lasting cura-
tive benefits, or – extrapolating the potential
of gene editing to its logical end – by simply
“cutting out” genetic diseases. However, as with
all medical advances, gene editing and gene
therapies also raise questions about ethics,
risks, affordability, and regulation. 2
“The new editing technology is a revolu-
tion for science and for medicine,” Stuart Orkin,
MD, associate chief of the division of hematol-
ogy/oncology and chairman of the pediatric
oncology department at Boston Children’s
Hospital and David G. Nathan Professor of Pe-
diatrics at Harvard Medical School, in Boston,
Massachusetts, told ASH Clinical News. “The
technology is being rapidly improved, and it is
highly likely that it will be applied for several
disorders in the near future, with positive
effect.”
So, what is CRISPR/Cas9, and what do
hematologists need to know about this tech-
nology as it transitions from the laboratory to
the clinic? ASH Clinical News spoke with Dr.
Orkin and other researchers specializing in
CRISPR/Cas9 gene editing for answers.
“Today, we are
limited more by
our imagination
and by figuring out
the right question
than by the tools at
hand.”
—MARGARET GOODELL, PhD
102
ASH Clinical News
Gene Editing 101
Gene editing (also known as genome editing)
refers to the alteration of DNA at specific
locations in the genome. 3 Several gene-editing
technologies have been developed, all based
on nucleases (enzymes that cleave nucleic
acids) that are delivered to targeted cells, then
recognize, bind, and cleave a target sequence
of DNA (see FIGURE 1 ).
Gene editing takes advantage of the cell’s
own DNA repair mechanisms, which can
result in many different molecular outcomes,
such as:
• non-homologous end joining, which
reunites the broken ends of DNA and often
results in small insertions or deletions, and
can lead to gene disruption 4
• homology-driven repair (HDR), which
aids in introducing novel DNA by
exploiting donor DNA molecules that have
homologous sequences surrounding the
DNA break 5
Meganucleases were the first targeted nucleases
to be used for gene editing. 4 More recently,
gene editing has been revolutionized by
nuclease-based technologies with improved
speed, cost, accuracy, and efficiency, including
zinc-finger nucleases (ZFNs) and transcription
activator-like effector nucleases (TALENs; see
FIGURE 2 ). 3
ZFNs
ZFNs are engineered DNA-binding proteins
that facilitate targeted genome editing by creat-
ing double-strand breaks in DNA at specified
locations. ZFNs consist of a FokI nuclease do-
main and three to six DNA-binding zinc-finger
domains. Because the FokI nuclease functions
as a dimer, a pair of ZFNs is engineered to bind
nine to 18 base pairs of DNA on either side of
the target sequence. Like meganucleases, ZFNs
require complicated engineering for each new
target DNA sequence. 4,5
TALENs
TALENs are also fusions of a FokI nuclease
domain and a DNA-binding domain
(transcription activator-like [TAL] proteins).
When two TALENs bind and meet, the
FokI domains create a double-strand break
that can “turn off ” a gene or can be used to
insert DNA. One advantage of TALENs over
ZFNs is their more straightforward, modular
engineering: Each TAL binds a single DNA
base, so they can be arranged in any order to
create novel DNA-binding domains. Like ZFNs,
specificity is increased due to the requirement
for dimerization of FokI. Recently, smaller
hybrid megaTALs have been created from
meganuclease plus TAL repeats. 4,5
CRISPR/Cas9
CRISPR/Cas9 is the most recent gene editing
tool to be added to the repertoire. It is adapted
from a naturally occurring genome editing sys-
tem in which bacteria capture snippets of DNA
from invading viruses and use them to create
DNA segments known as CRISPR arrays. The
CRISPR arrays allow the bacteria to “remem-
ber” the invading viruses (or closely related
ones). If they invade again, the bacteria produce
RNA segments from the CRISPR arrays to
target the viruses’ DNA. The bacteria use Cas9
or a similar enzyme to specifically cleave the
viruses’ DNA, which disables the virus. 3
In the CRISPR/Cas9 technology most of-
ten used for gene editing, a single guide RNA
(sgRNA) directs Cas9 to cleave at a specific
site – essentially cutting out and shutting off
the targeted gene.
Scientists can create a new sgRNA for any
genomic target, and the Cas9 nuclease cut-
ting tool can be used with multiple sgRNAs
to make multiple changes simultaneously.
CRISPR/Cas9’s use of RNA as a reagent offers
a major advantage over ZFNs and TALENs,
which require complicated and expensive pro-
tein engineering for each new target. 4,5
“CRISPR is easy, efficient, and relatively
inexpensive. This is such an accessible tech-
nology that we have an entire new toolkit in
the community,” said Margaret Goodell, PhD,
a professor at Baylor College of Medicine
and director of the Stem Cell and Regenera-
tive Medicine Center in Houston, Texas. “The
pace of experiments and the types of experi-
ments that we can now do has fundamentally
changed. Today, we are limited more by our
imagination and by figuring out the right
question than by the tools at hand.”
Ethical Considerations of Gene Therapy
Gene therapy refers to the modification of a
dysfunctional gene to treat or cure disease.
Today’s gene-editing technologies can be used
for g ene therapies, which fall into the follow-
ing two categories: 3
• somatic therapies alter the DNA in non-
reproductive cells so changes affect only
the person receiving therapy
• germline therapies alter the DNA in
reproductive cells so changes can be passed
down to future generations
December 2017