Genome editing

Genome editing, or genome editing with engineered genetic information and an exogenous DNA. These
nucleases (GEEN) is a type of genetic engineering methods have been made possible in yeast and mice.
in which DNA is inserted, deleted or replaced in the
genome of a living organism using engineered nucleases, These approaches have several drawbacks:
or molecular scissors. These nucleases create site-
specic double-strand breaks (DSBs) at desired locations PCR- and phage-mediated approaches are less suc-
in the genome. The induced double-strand breaks are cessful in more complex organisms such as mam-
repaired through nonhomologous end-joining (NHEJ) or mals, where delivery becomes more dicult.
homologous recombination (HR), resulting in targeted
mutations ('edits). They also require stringent selection steps and thus
the addition of selection-specic sequences, along
As of 2015 there were four families of engineered nu- with those incorporated into the DNA.
cleases being used: meganucleases, zinc nger nucleases
(ZFNs), transcription activator-like eector-based nucle- Recombination-based methods can be quite ine-
ases (TALEN), and the CRISPR-Cas system.[1][2][3][4] cient - e.g. in mouse embryonic stem cells treated
The structure of 9 genome editors as of 2017 can be with donor DNA, only 1 in a million DNA molecules
viewed.[5] was incorporated at the desired position.[10]
Genome editing was selected by Nature Methods as the
The use of other mutagenic techniques (such as P-
2011 Method of the Year.[6] The CRISPR-Cas system
element transgenesis in Drosophila) also has limitations,
was selected by Science as 2015 Breakthrough of the
[7] the major one being the randomness of incorporation and
Year.
the possibility of aecting other genes and expression
patterns.
Hence, genome editing with engineered nucleases is a
1 Concept promising new approach. This rapidly evolving tech-
nology overcomes these shortcomings and uses relatively
A common approach in modern biological research is to simple concepts.
modify the DNA sequence (genotype) of an organism (or
a single cell) and observe the impact of this change on the
organism (phenotype). This approach is called reverse 1.1 Double stranded breaks and their re-
genetics and its signicance for modern biology lies in its pair
relative simplicity. This method contrasts with that of
forward genetics, where a new phenotype is rst observed Fundamental to the use of nucleases in genome editing
and then its genetic basis is studied. This course is more is the concept of DNA double stranded break (DSB) re-
complex because phenotypic changes are often a result of pair mechanics. One of the known DSB repair pathways
multiple genetic interactions. that are essentially functional in all organisms are the non-
homologous end joining (NHEJ) and homology directed
Among the key requirements of reverse genetic analysis
repair (HDR).
is the ability to modify the DNA sequence of the target
organism. This can be achieved by: NHEJ uses a variety of enzymes to directly join the DNA
ends in a double-strand break. In contrast, in HDR, a ho-
mologous sequence is utilized as a template for regener-
site-directed mutagenesis employing either phage-
ation of missing DNA sequence at the break point. The
or polymerase chain reaction (PCR)-mediated
natural properties of these pathways form the very basis
methods and oligonucleotides containing the desired
[8] of nuclease-based genome editing.
mutation. These methods are most useful in or-
ganisms with straightforward methods for the intro- NHEJ is error-prone, and has been shown to cause muta-
duction and selection of genes of interest, such as tions at the repair site in approximately 50% of DSB in
bacteria and yeast.[9] mycobacteria,[11] and also its low delity has been linked
to mutational accumulation in leukemias.[12] Thus if one
recombination based methods that utilize the natural is able to create a DSB at a desired gene in multiple sam-
ability of cells to exchange DNA between its own ples, it is very likely that mutations will be generated at

1
2 1 CONCEPT

that site in some of the treatments because of errors cre-

ated by the NHEJ indelity.
On the other hand, the dependency of HDR on a homolo-
gous sequence to repair DSBs can be exploited by insert-
ing a desired sequence within a sequence that is homol-
ogous to the anking sequences of a DSB which, when
used as a template by HDR system, would lead to the
creation of the desired change within the genomic region
of interest.
Despite the distinct mechanisms, the concept of the HDR
based gene editing is in a way similar to that of homolo-
gous recombination based gene targeting. However, the
rate of recombination is increased by at least three orders
of magnitude when DSBs are created and HDR is at work
thus making the HDR based recombination much more
ecient and eliminating the need for stringent positive
and negative selection steps.[13] So based on these prin-
ciples if one is able to create a DSB at a specic location Groups of engineered nucleases used for GEEN. Matching colors
within the genome, then the cells own repair systems will signify DNA recognition patterns
help in creating the desired mutations.

signed meganuclease (US Patent 8,021,867 B2).

1.2 Site-specic double stranded breaks
Creation of a DSB in DNA should not be a challenging
task as the commonly used restriction enzymes are capa-
ble of doing so. However, if genomic DNA is treated with
a particular restriction endonuclease many DSBs will be
created. This is a result of the fact that most restriction
enzymes recognize a few base pairs on the DNA as their
target and very likely that particular base pair combina-
tion will be found in many locations across the genome.
To overcome this challenge and create site-specic DSB,
three distinct classes of nucleases have been discovered
and bioengineered to date. These are the Zinc nger nu-
cleases (ZFNs), transcription-activator like eector nu-
cleases (TALEN) and meganucleases. Below is a brief
overview and comparison of these enzymes and the con-
cept behind their development.
1.3 Engineered nucleases
Meganucleases, found commonly in microbial species,
have the unique property of having very long recogni-
tion sequences (>14bp) thus making them naturally very
specic.[14][15] This can be exploited to make site-specic
DSB in genome editing; however, the challenge is that
not enough meganucleases are known, or may ever be
known, to cover all possible target sequences. To over-
come this challenge, mutagenesis and high throughput
screening methods have been used to create meganucle-
ase variants that recognize unique sequences.[15] Others
have been able to fuse various meganucleases and cre-
ate hybrid enzymes that recognize a new sequence.[16]
Yet others have attempted to alter the DNA interacting
aminoacids of the meganuclease to design sequence spe-
cic meganucelases in a method named rationally de-
Meganucleases have the benet of causing less toxicity
in cells than methods such as ZFNs, likely because of
more stringent DNA sequence recognition;[15] however,
the construction of sequence-specic enzymes for all pos-
sible sequences is costly and time consuming, as one is not
beneting from combinatorial possibilities that methods
such as ZFNs and TALEN-based fusions utilize.
As opposed to meganucleases, the concept behind ZFNs
and TALEN technology is based on a non-specic DNA
cutting enzyme, which can then be linked to specic
DNA sequence recognizing peptides such as zinc ngers
and transcription activator-like eectors (TALEs).[17]
The key to this was to nd an endonuclease whose DNA
recognition site and cleaving site were separate from each
other, a situation that is not common among restriction
enzymes.[17] Once this enzyme was found, its cleaving
portion could be separated which would be very non-
specic as it would have no recognition ability. This por-
tion could then be linked to sequence recognizing pep-
tides that could lead to very high specicity. A restriction
enzyme with such properties is FokI. Additionally FokI
has the advantage of requiring dimerization to have nu-
clease activity and this means the specicity increases
dramatically as each nuclease partner would recognize
a unique DNA sequence. To enhance this eect, FokI
nucleases have been engineered that can only function
as heterodimers and have increased catalytic activity.[18]
The heterodimer functioning nucleases would avoid the
possibility of unwanted homodimer activity and thus in-
crease specicity of the DSB. Although the nuclease por-
tions of both ZFNs and TALEN constructs have simi-
lar properties, the dierence between these engineered
nucleases is in their DNA recognition peptide. ZFNs
rely on Cys2-His2 zinc ngers and TALEN constructs
on TALEs. Both of these DNA recognizing peptide do-
2.1 Targeted gene modication in plants 3

mains have the characteristic that they are naturally found
in combinations in their proteins. Cys2-His2 Zinc n-
gers typically happen in repeats that are 3 bp apart and
are found in diverse combinations in a variety of nu-
cleic acid interacting proteins such as transcription fac-
tors. TALEs on the other hand are found in repeats with
a one-to-one recognition ratio between the amino acids
and the recognized nucleotide pairs. Because both zinc
ngers and TALEs happen in repeated patterns, dier-
ent combinations can be tried to create a wide variety of
sequence specicities.[14] Zinc ngers have been more es-
tablished in these terms and approaches such as modular
assembly (where Zinc ngers correlated with a triplet se-
quence are attached in a row to cover the required se-
quence), OPEN (low-stringency selection of peptide do-
mains vs. triplet nucleotides followed by high-stringency
selections of peptide combination vs. the nal target in
bacterial systems), and bacterial one-hybrid screening of
zinc nger libraries among other methods have been used
to make site specic nucleases.
1.4 Precision of engineered nucleases
Because o-target activity of an active nuclease would
have potentially dangerous consequences at the genetic
and organismal levels, the precision of meganucleases,
ZFNs, CRISPR, and TALEN-based fusions has been
an active area of research. While variable gures have
been reported, ZFNs tend to have more cytotoxicity
than TALEN methods or RNA-guided nucleases, while
TALEN and RNA-guided approaches tend to have the
greatest eciency and fewer o-target eects.[19] Based
on the maximum theoretical distance between DNA
binding and nuclease activity, TALEN approaches result
in the greatest precision.[4]
becoming a standard experimental strategy in research
labs.[20] The recent generation of rat, zebrash, maize and
tobacco ZFN-mediated mutants and the improvements in
TALEN-based approaches testify to the signicance of
the methods, and the list is expanding rapidly. Genome
editing with engineered nucleases will likely contribute
to many elds of life sciences from studying gene func-
tions in plants and animals to gene therapy in humans.
For instance, the eld of synthetic biology which aims to
engineer cells and organisms to perform novel functions,
is likely to benet from the ability of engineered nuclease
to add or remove genomic elements and therefore create
complex systems.[20] In addition, gene functions can be
studied using stem cells with engineered nucleases.
Listed below are some specic tasks this method can
carry out:
Targeted gene mutation
Creating chromosome rearrangement
Study gene function with stem cells
Transgenic animals
Endogenous gene labeling
Targeted transgene addition

2 Applications

Overview of GEEN workow and editing possibilities

2.1 Targeted gene modication in plants

Genome editing using meganucleases,[21] ZFNs, and
TALEN provides a new strategy for genetic manipula-
tion in plants and are likely to assist in the engineer-
ing of desired plant traits by modifying endogenous
genes. For instance, site-specic gene addition in ma-
jor crop species can be used for 'trait stacking' whereby
several desired traits are physically linked to ensure
their co-segregation during the breeding processes.[18]
As of 2012 ecient genome editing had been developed Progress in such cases have been recently reported in
for a wide range of experimental systems ranging from Arabidopsis thaliana[22][23][24] and Zea mays. In Ara-
plants to animals, often beyond clinical interest, and was bidopsis thaliana, using ZFN-assisted gene targeting, two
4 3 PROSPECTS AND LIMITATIONS

herbicide-resistant genes (tobacco acetolactate synthase apy (ICAAC) held in Chicago from September 1720,
SuRA and SuRB) were introduced to SuR loci with as 2011.[34] Researchers at SGMO mutated CCR5 in CD4+
high as 2% transformed cells with mutations.[25] In Zea T cells and subsequently produced an HIV-resistant T-cell
mays, disruption of the target locus was achieved by ZFN- population.[35]
induced DSBs and the resulting NHEJ. ZFN was also Gene editing is used to generate modied custom im-
used to drive herbicide-tolerance gene expression cassette mune cells. For example, recent report indicated that T
(PAT) into the targeted endogenous locus IPK1 in this cells could be modied to inactivate the glucocorticoid
case.[26] Such genome modication observed in the re- receptor; the resulting immune cells are fully func-
generated plants has been shown to be inheritable and was
tional but resistant to the eects of commonly used
transmitted to the next generation.[26] corticosteroids.[36] Similarly, scientists at Cellectis re-
In addition, TALEN-based genome engineering has been cently generated custom T-cells expressing chimeric anti-
extensively tested and optimized for use in plants.[27] gen receptors using TALEN technology.[37] These T-cells
TALEN fusions have also been used to improve the qual- can be engineered to be resistant to anti-cancer drugs and
ity of soybean oil products[28] and to increase the storage to invoke immune responses against targets of interest.[38]
potential of potatoes[29] The rst clinical use of TALEN-based genome edit-
Several optimizations need to be made in order to ing was in the treatment of CD19+ acute lymphoblastic
improve editing plant genomes using ZFN-mediated leukemia in an 11-month old child.[39] Modied donor
targeting.[30] These include the reliable design and sub- T cells were engineered to attack the leukemia cells, to
sequent test of the nucleases, the absence of toxicity of be resistant to Alemtuzumab, and to evade detection by
the nucleases, the appropriate choice of the plant tissue the host immune system after introduction. A few weeks
for targeting, the routes of introduction or induction of after therapy, the patients condition improved. Though
enzyme activity, the lack of o-target mutagenesis, and a physicians are cautious, the patient has been in remission
reliable detection of mutated cases.[30] for several months following treatment.[40][41]

2.2 Gene therapy

3 Prospects and limitations
The ideal gene therapy practice is that which replaces the
defective gene with a normal allele at its natural location.
This is advantageous over a virally delivered gene as thereIn the future, an important goal of research into genome
is no need to include the full coding sequences and reg- editing with engineered nucleases must be the improve-
ulatory sequences when only a small proportions of the ment of the safety and specicity of the nucleases. For
gene needs to be altered as is often the case.[31] The ex- example, improving the ability to detect o-target events
pression of the partially replaced genes is also more con- can improve our ability to learn about ways of preventing
sistent with normal cell biology than full genes that are them. In addition, zinc-ngers used in ZFNs are seldom
carried by viral vectors. completely specic, and some may cause a toxic reac-
tion. However, the toxicity has been reported to be re-
Gene targeting through ZFNs or TALEN-based ap- duced by modications done on the cleavage domain of
proaches can also be used to modify defective genes the ZFN.[31]
at their endogenous chromosomal locations. Exam-
ples include the treatment of X-linked severe com- In addition, research by Dana Carroll into modifying the
genome with engineered nucleases has shown the need for
bined immunodeciency (X-SCID) by ex vivo gene cor-
rection with DNA carrying the interleukin-2 receptor better understanding of the basic recombination and re-
pair machinery of DNA. In the future, a possible method
common gamma chain (IL-2R)[32] and the correction
of Xeroderma pigmentosum mutations in vitro using to identify secondary targets would be to capture broken
TALEN.[33] Insertional mutagenesis by the retroviral vec- ends from cells expressing the ZFNs and to sequence [31]
the
tor genome induced leukemia in some patients, a prob- anking DNA using high-throughput sequencing.
lem that is predicted to be avoided by these technologies. Genome editing occurs also as a natural process without
However, ZFNs may also cause o-target mutations, in articial genetic engineering. The agents that are com-
a dierent way from viral transductions. Currently many petent to edit genetic codes are viruses or subviral RNA-
measures are taken to improve o-target detection and agents.[42]
ensure safety before treatment. Although GEEN has higher eciency than many other
Recently, Sangamo BioSciences (SGMO) introduced the methods in reverse genetics, it is still not highly ecient;
Delta 32 mutation (a suppressor of CCR5 gene which is in many cases less than half of the treated populations
a co-receptor for HIV-1 entry into T cells therefore en- obtain the desired changes.[25] For example, when one is
abling HIV infection) using Zinc Finger Nuclease (ZFN). planning to use the cells NHEJ to create a mutation, the
Their results were presented at the 51st Interscience cells HDR systems will also be at work correcting the
Conference on Antimicrobial Agents and Chemother- DSB with lower mutational rates.
 5

3.1 Human enhancement who might use the knowledge to create vaccine resistant
strains of other pox viruses, such as smallpox, that could
Many transhumanists see genome editing as a potential aect humans.[48] Furthermore, there are additional con-
tool for human enhancement.[43][44][45] Australian biol- cerns about the ecological risks of releasing gene drives
ogist and Professor of Genetics David Andrew Sinclair into wild populations.[48][54][55]
notes that the new technologies with genome editing will
allow it to be used on individuals [...] to have [...] health-
ier children - designer babies.[46] According to a Septem- 5 See also
ber 2016 report by the Nueld Council on Bioethics in
the future it may be possible to enhance people with genes
Genome engineering
from other organisms or wholly synthetic genes to for ex-
[47][48]
ample improve night vision and sense of smell. Germinal choice technology
The American National Academy of Sciences and
National Academy of Medicine issued a report in Febru- CRISPR/Cpf1
ary 2017 giving qualied support to human genome TALEN
editing.[49] They recommended that clinical trials for
genome editing might one day be permitted once answers NgAgo, a ssDNA-guided Argonaute endonuclease
have been found to safety and eciency problems but
only for serious conditions under stringent oversight.[50]
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[39] Pollack, Andrew (2015-11-05). A Cell Therapy
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8 8 TEXT AND IMAGE SOURCES, CONTRIBUTORS, AND LICENSES

8 Text and image sources, contributors, and licenses

8.1 Text
Genome editing Source: https://en.wikipedia.org/wiki/Genome_editing?oldid=766545858 Contributors: Bearcat, Ceyockey, Kell, Joe
Roe, Rjwilmsi, Twr57, MaxEnt, Headbomb, Smartse, Magioladitis, Hifrommike65, BatteryIncluded, R'n'B, Katharineamy, SylviaStan-
ley, Malcolmxl5, Sunrise, Excirial, Galapah, Jytdog, Stevenmcrane, Yobot, AnomieBOT, Bellerophon, A412, DadOfBeanAndBug, Mean
as custard, Lopifalko, Bamyers99, ClueBot NG, BG19bot, CitationCleanerBot, ArticlesForCreationBot, David.moreno72, Seahorsechip-
munk, Anomie111, Sidsandyy, CuriousMind01, Me, Myself, and I are Here, N1K0W1N, Evolution and evolvability, KramerAsh, Fixuture,
Vpliatsika, GenomeEditor, Crystallizedcarbon, Dcbennett2, InternetArchiveBot, Eno Lirpa, Bender the Bot, Tyree999, DennisPietras and
Anonymous: 21

8.2 Images
File:Endogenous_genes_targeted.jpg Source: https://upload.wikimedia.org/wikipedia/en/0/02/Endogenous_genes_targeted.jpg Li-
cense: CC-BY-SA-3.0 Contributors:
microsoft word
Original artist:
Seahorsechipmunk
File:Engineered_Nucleases.jpg Source: https://upload.wikimedia.org/wikipedia/en/c/cd/Engineered_Nucleases.jpg License: CC-BY-
SA-3.0 Contributors:
Powerpoint
Previously published: No
Original artist:
Farzad Jamshidi
File:Lock-green.svg Source: https://upload.wikimedia.org/wikipedia/commons/6/65/Lock-green.svg License: CC0 Contributors: en:File:
Free-to-read_lock_75.svg Original artist: User:Trappist the monk
File:Merge-arrows.svg Source: https://upload.wikimedia.org/wikipedia/commons/5/52/Merge-arrows.svg License: Public domain Con-
tributors: ? Original artist: ?
File:Possibilities_of_genome_editing.jpg Source: https://upload.wikimedia.org/wikipedia/en/0/07/Possibilities_of_genome_editing.
jpg License: CC-BY-SA-3.0 Contributors:
Powerpoint
Previously published: No
Original artist:
Farzad Jamshidi

8.3 Content license

Creative Commons Attribution-Share Alike 3.0