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Engineered Nucleases
William Clinton Welsh
Engineered Nucleases:
A Novel Tool in Genetic Modification Research
Introduction of Topic
An important part of gene modification and gene therapy is the ability to recognize a
specific sequence of DNA. Most forms of gene modification involve breaking the DNA and
then modifying the damaged site, possibly with new genetic material (Pan et al. 2012).
Specificity means that the break occurs only where you want it to occur, which allows you
to control where in an organism’s genome the modification takes place as well as minimize
unwanted modifications. To facilitate these breaks, a DNA cleaving enzyme known as a
nuclease is used. The development of engineered nucleases that can be customized for
specific sequences and higher selectivity is now allowing for the research of new forms of
treatment and gene therapy. This paper focuses on two particular engineered nucleases,
the zinc-finger nuclease (ZFN) and the transcription activator-like effector nuclease (TALEN),
and some of the research that has been conducted to test the potential of these nucleases
for use in novel forms of treatment and therapy.
Important Terms
Nuclease
A nuclease is an enzyme that will cleave DNA by hydrolyzing the phosphodiester
backbone. Most nucleases will cleave only one strand of DNA at a time, such as those
involved in DNA repairs that require only a single base or chain of bases to be removed.
Nucleases are further divided into exonucleases and endonucleases. Exonucleases will
cleave nucleotides one at a time, starting at one end of the DNA strand, until the entire
strand is degraded. Endonucleases will cleave DNA somewhere in the middle of the strand
and the most common kinds are called restriction nucleases. Restriction Nucleases are
capable of recognizing a specific sequence of DNA, and will cleave only at a particular site
(known as a restriction site), and will typically cleave DNA across both strands. Other types
of Endonucleases are non-specific, and can either cleave the DNA anywhere, or will rely on
a DNA binding domain that is separate from the nuclease for site recognition. The latter
will be the case with zinc-finger nucleases and TALE nucleases, which will be discussed in a
later section of this paper.
Homologous recombination and Non-homologous End Joining (Mao et al. 2008)
There are two DNA repair pathways that are central to the types of gene
modification that are discussed in this paper: the Homologous recombination pathway and
non-homologous end-joining pathway. Both of these pathways come into play when the cell
needs to repair a double strand break; a break across both DNA strands. These pathways
are exploited in ways that make gene modification possible. How this is done is discussed
later in the paper, but it is first important to know how these pathways typically function.
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The primary difference between these two pathways is that homologous
recombination relies on homology, whereas non-homologous end joining does not. Recall
that Homology refers to having two copies of a gene on separate, homologous
chromosomes. A cell can have homologous chromosomes when it replicates its genome for
cell division or will naturally carry two copies at all times, such is the case with diploid
cells. Amongst mammalian and other sexually reproducing diploid organisms, one
chromosome is paternally inherited and the other is maternally inherited.
When a double-stranded break occurs in the cell, the cell will primarily utilize Nonhomologous End Joining, which, as its name implies, is when the cell repair DNA by repairing
the broken ends. The rate at which cells utilize non-homologous end joining is significantly
higher that the rate of homologous recombination (Mao et al. 2008). The results, however,
are less than ideal. If the break results in a hanging end, or sticky end, in which one or
more unpaired nucleotide bases trail off the broken end, the repair process will first need
to remove those bases before it can reattach the DNA strands. Since there is no template to
use for comparison, non-homologous end joining will result in a loss of genetic information
when those nucleotide bases are removed.
Homologous Recombination differs from non-homologous end joining in a number of
ways. When a double stranded break occurs, one of the DNA strands on the broken end can
attach to a separate complimentary strand on the respective homologous chromosome.
The homologous recombination pathway will then use the homologous chromosome as a
template to repair the damage DNA strand. Since a template is used, homologous
recombination typically results in complete fidelity.
As mentioned, these are pathways, meaning that each repair involves a series of
steps and multiple enzymes and other factors. The following diagram illustrates simplified
versions of the two repair pathways (homologous recombination is abbreviated HR, and
non-homologous end joining is NHEJ):
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William Clinton Welsh
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William Clinton Welsh
Gene Therapy
Gene Therapy entails the use of gene modification as a means to treat disease. Gene
therapy offers a cure for diseases that conventional medicine cannot cure, such as genetic
disorders, or can offer alternative methods to treat diseases that prove difficult for
conventional medicine, such as cancer and certain viral infections. There are two types of
gene therapy:
Forward/Traditional
Forward, or Traditional Gene Therapy corrects a faulty gene. The faulty gene is
removed, and a corrected gene or normal gene is substituted in place.
Reverse (Pan et al. 2012)
Reverse Gene Therapy aims at destroying or altering the function of a gene involved
in the mechanics of a disease. A mutation is induced that will: Suppress an overexpressed
gene, destroy the formation of a disease causing protein, alter a surface protein necessary
for certain viral infections, etc.
Viral Vectors
Viruses, particularly retroviruses, are used as a delivery vehicle that brings the gene
modifying mechanisms to the target cells. Because these viruses naturally have the
infrastructure in place to infect cells with foreign genes, and even have those genes
integrate into the host’s genome, they are well-suited to be reengineered and utilized for
gene modification, and are referred to as Viral Vectors. Viral Vectors are not discussed in
this paper, but it is important to understand that they are a common tool for bringing the
nucleases and other gene modifying factors to the cells of an organism.
Embryonic Stems Cells vs. Induced Pluripotent Stem Cells (Zou et al. 2009)
Embryonic Stems Cells are pluripotent stem cells derived from an early-stage
embryo (undifferentiated). Induced Pluripotent Stem Cells are derived from adult somatic
cells (differentiated) that have been induced to return to a pluripotent state, and behave
similar to Embryonic Stem Cells.
Structure of ZFN and TALEN
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ZFN and TALEN are fairly similar to each other in composition, each containing two
major domains: a nuclease domain and a DNA binding domain (see diagram above). The
nuclease domain consists of a FokI restriction enzyme (Pan et al. 2012), which will cleave
DNA non-specifically on its own (David), but is given specificity by an attached DNA
binding domain. The DNA binding domain for ZFN consists of a series of repeated zinc
finger subunits, and the DNA binding domain for TALEN consists of a series of repeated
transcription activator-like effectors (TALEs) subunits (Pan et al. 2012). Each of the
subunits in the DNA binding domains have the ability to recognize a specific nucleotide or
set of nucleotides, with each zinc finger able to recognize a specific set of 3 nucleotides and
each TALE able to recognize one specific nucleotide (Pan et al. 2012). By modifying the
arrangement of these subunits, the nuclease can be customized to recognize different
sequences of DNA.
Function of ZFN and TALEN in Gene Modification
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ZFNs and TALENs have been used successfully in three types of gene modification:
Gene Addition, Gene Correction, and Gene Disruption (Pan et al. 2012).
Both Gene Addition and Gene Correction take advantage of the homologous
recombination pathway. Recall that homologous recombination requires the presence of a
homologous template. In Gene Addition, the DNA template is a gene that is new to the
genome. When this new gene sequence is used by the cell as a homologous template for
homologous recombination, this will result in an addition to the genome. In Gene
Correction, the DNA template is a correct or normal counterpart to a faulty gene in the
genome. When used by the cell as a homologous template, the DNA template acts as a
‘spell-check’ and allows the cell to correct the mistake presently in its genome. Both Gene
Addition and Gene Correction are similar in that the mechanisms used are the same, and
are different in that the purpose for Addition is to add something novel to the genome and
Correction is to correct a mistake already present in the genome.
Gene Disruption is vastly different from Gene Addition and Correction. No DNA
template is needed, because Gene Disruption can take advantage of the non-homologous
end-joining pathway. Recall that if a DNA break results in a hanging, or sticky end, and the
cell uses the non-homologous end joining pathway, the hanging nucleotide base pair must
first be degraded before the ends can be attached, which will result in the loss of genetic
information. If this form of deleterious repair occurs in a gene, that gene can potentially be
crippled or mutated, and this is the intent of Gene Disruption. For Gene Disruption to work,
all that is needed is a nuclease that will cleave the DNA strand in the middle of a critical
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region (e.g. a promoter, enhancer, or gene) in such a way as to produce a hanging end.
Then the cells own repair pathway will degrade the hanging end. Even the loss of a single
nucleotide base pair will result in a frameshift mutation that can completely shut down a
gene, making Gene Disruption a simpler method for gene modification.
Experimental Application and Analysis of Engineered Nucleases
The Use of Zinc Finger Nuclease in Gene Targeting in Human Embryonic and Induced
Pluripotent Stem Cells
-A discussion of the research conducted by Zou et al. (2009)
Embryonic and Induced Pluripotent Stem Cells have the potential to lead to highly
beneficial stem cell therapies once effective gene targeting methods are developed. These
gene-targeting methods rely on homologous recombination repair, and were known to
have low efficiency amongst human embryo stem cells during the time this research was
conducted. There had also been no studies conducted to determine the effectiveness of
gene targeting on induced pluripotent stem cells. The potential of ZFN’s to increase the
efficiency of gene targeting prompted researchers to conduct this study.
Researchers first developed an Enhanced Green Fluorescent Protein (EGFP) reporter
gene in order to determine whether gene modification occurred. This gene, in its normal
state, would code for a protein that would fluoresce green under observation. Next, the
researchers modified this gene so that it would be blocked by another sequence that would
halt the translation of the protein. Using a viral vector, the suppressed EGFP gene was
inserted into a population of embryonic cells and a population induced pluripotent cells.
The genetic sequence containing the suppressed EGFP gene also contained a gene that
conferred antibiotic resistance to puromycin, which allowed researchers to use puromycin
to select only for cells that successfully incorporated the full insert. Next, ZFN’s that
targeted the sequence suppressing the EGFP and donor DNA coding for an unsuppressed
EGFP were administered to the cell populations, and the return of green fluorescence was
used to gauge the effectiveness of the treatment (see below, human embryonic cells on the
left, and induced pluripotent on the right).
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William Clinton Welsh
The efficiency of this treatment was (.24%) in human embryonic cells and (.14%) in
induced pluripotent stem cells, and no unwanted modification could be detected. Though
the proportion of differentiated cells is low, this paper demonstrates the potential uses of
ZFN’s in gene targeting, and the effectiveness of these types of engineered nucleases will
improve with time. Human embryo and induced pluripotent stem cells were shown to be
modifiable by an engineered nuclease, an important finding which will help lead to more
research in the development of new kinds of stem cell therapies.
Optimizing Transcription Activator-Like Effector Nucleases
-A discussion of the research conducted by Ning et al. (2012)
The researchers were able to construct a TALEN that could recognize the sequence
in the Human β-globin (HBB) gene responsible for sickle cell anemia. However, in order to
do so, the researchers had to first address some underlying structural problems inherit to
ZFN and TALEN. By modifying the subunits that compose the DNA binding domain,
researchers were able to develop a more optimal form of TALEN.
Though ZFN’s are widely used, modifying the zinc finger subunits proves to be
incredibly difficult; each subunit must recognize a set of 3 nucleotides, which makes it
structurally challenging to modify, and can result in the need for a large variety of unique
subunits. What is more, the zinc finger subunits have low binding affinity if not properly
design, and the lack of specificity may result in off-target site cleavages. Since each TALE
subunit on the TALEN only recognizes one nucleotide, the number of necessary
combinations is smaller (4 instead of potentially 64). TALE’s are also structurally easier to
modify. Each TALE domain consists of 33-35 amino acids, but recognition occurs only at
the 12th and 13th amino acid. By changing the combination of just these two amino acids,
TALE’s can be made to recognize different nucleotide bases. While TALEN’s easier to work
with than ZFN’s, they come with their own difficulties.
There are two unique features regarding the structure and function of TALEN’s:
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William Clinton Welsh
Notice that the DNA binding domains (denoted as gray bars) have a Thymine (T) at the 5’
position (denoted by green circles), and that each end of the binding domain has an
extension of amino acids (denoted by the white squares). The length of these end
extensions, and whether there is a thymine at the 5’ position, can greatly affect the cleaving
efficiency of the TALEN, and tests were conducted to determine how this response could be
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used to optimize TALEN. These tests were conducted by using yeast with
a mutated
lacZ
gene and homologous gene repair as a means of determining the efficiency of the TALEN’s:
NG or
DNA
etween
ence of
and its
ructing
apeutic
eavage
erminal
spacer
of the
AL EN
BEs not
esigned
B gene
caffolds
hanced
oxicity.
correct
owerful
py.
ryzae27
Fig. 2 Schematic of the yeast reporter system. Details are provided in
the text. Recovered L acZ gene is shown in dotted box.
To determine the in vivo cleavage efficiency of a TA L EN , we
established a yeast reporter system adapted from a previously
reported single strand annealing assay.29 The reporter plasmid
contains a divided lacZ gene in which a duplicated 100 bp
portion of the lacZ coding region has been created. The two
truncated lacZ D N A fragments are separated by a PTS
containing an in-frame stop codon between the two EBE sites.
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William Clinton Welsh
First the researchers varied the N-terminal and C-terminal ends of the TALE:
(N#) describes the length of the N-terminal, (C#) describes the length of the C-terminal,
and (#bp) describes the length of the DNA segment being cleaved. I-Crel is a different
restriction endonuclease that was used as a control for comparison. The black dots
describe the activity of the nuclease (ND standing for ‘no detectable activity’). Researchers
started by modifying the N-terminal. When N3 was determined to be the most optimal,
they then preceded to modify the C-terminal while keeping the N-terminal constant at N3.
Next, tests were conducted to determine how these TALEN’s of varying length
responded to different nucleotide pairs at the 5’ position:
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William Clinton Welsh
The five most optimal length combinations were tested against each of the nucleotide
bases. TALE prefers thymine (T) to be at the 5’ location, and this preference is shown by
the data. However, by varying the length of the C-terminal (the terminal in contact with the
5’ nucleotide), the researchers were able to develop a TALEN that could function with
greater efficiency even without a 5’ thymine (denoted by the red circle).
The success of novel forms of treatment involving gene therapy, gene modification,
and stem cell therapy depends heavily on the efficiency of mechanisms performing the
modification. By optimizing TALEN, the researchers were later able to develop a TALEN
that could recognize the sequence for the mutated HBB gene responsible for sickle cell
anemia (which is discussed in the article, but won’t be discussed in this paper). It is
important to understand that these engineered nucleases are dynamic creations; the forms
of each component can be tweaked and modified to enhance their overall performance, and
finding the perfect balance will be what brings the novel treatments into actuality.
Genetic Disruption of Hepatitis B
-A discussion of the research conducted by Bloom et al. (2013)
The implications that nucleases have on improving human health are not just
limited to modifying the human genome. As the research in this article shows, human
health can be improved by attacking the genomes of pathogens such as Hepatitis. So far,
engineered nucleases have been shown to be beneficial influences in the development of
genetic treatments. Their high selectivity for only certain sequences makes then less toxic
to cells than other, less selective restriction enzymes. However, this selectivity also makes
then an ideal weapon. Here we have TALEN’s that have been engineered to target
conserved sequences in the genome of the Hepatitis B virus (HBV). The black arrows
denote the recognition sites of the four TALEN’s:
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William Clinton Welsh
Four TALEN’s were developed, however, only the TALEN’s targeting the C and S sites
effectively disrupted HBV. The C site contains a gene important for the formation of the
viral core, and the S site contains a gene important for the formation of the surface protein
HBsAg. The effectiveness of these TALEN’s was determined by a test run on HBV-infected
mice:
Graphs A depicts the concentration of HBsAg in blood serum extracted from mice treated
with TALEN-S, TALEN-C, and no TALEN treatment. Graph B shows concentrations of
circulating viral particles (VPE’s). Graph C shows the ratio of HBV mRNA to GAPDH mRNA
(a naturally occurring gene). Graph D represents the concentration of HBV core protein
positive cells.
While the results show that the use of TALEN’s did not cure the mice, they do reveal
that HBV could be suppressed by using TALEN’s. Used in conjunction with other forms of
treatment, TALEN’s have the potential to improve the well-being of HBV positive
individuals. Here we see that TALEN’s can be used in an entirely different way, as a
selective toxin. This study illustrates the fact that these engineered nucleases are not
benign. If it weren’t for their high selectivity, these nucleases would be incredibly
cytotoxic, because nucleases are enzymes that degrade DNA by design. However, since
they can be given the ability to distinguish only the most precise sequences, the danger that
they pose to cells can instead be a blessing.
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