Marielle Alyanna T.

Bangalan
2012-27186

Genome Editing

Background

Genome editing has been considered as an invaluable research tool because of its vast

potential in terms of application in human therapies, microbial bioengineering, and agricultural

biotechnology. Using an engineered nuclease, a double-strand break is introduced on the

desired loci in the genome. Afterwards, this double-strand break initiates the cellular DNA repair

mechanism via nonhomologous end joining (NHEJ) or homology-directed repair (HDR).

At present, four major classes of nucleases namely (1) meganucleases and their other

derivatives such as (2) zinc-finger nucleases (ZFNs), (3) transcription activator-like effector

nucleases (TALENs), and (4) clustered regularly-interspaced short palindromic repeats

(CRISPR/Cas9) system have been used to enable site-specific genome editing. These

techniques will be further explored in this review paper.

History

In 1972, Paul Berg, a professor at Stanford University, assembled the first recombinant

DNA by isolating, cutting, and combining genes from various organisms. His study laid the

foundations of genetic engineering and the subsequent developments in recombinant DNA

(Baltimore et al., 2015). Following a series of advancements in techniques in biochemistry, he

was able to isolate and insert individual genes into rapidly growing organisms such as bacteria

or mammalian cells. The popularity of the restriction enzymes that acted as scissors to excise

specific DNA molecules and the discovery of DNA ligase that could piece spliced DNA together
were crucial to his discovery (Baltimore et al., 2015). Because of his significant contributions in

the field of genetic engineering, many scientists became interested in studying genetics and

gene editing. Following the success of his research, many controversies regarding the potential

dangers of recombinant DNA studies led to the development of National Academy’s Moratorium

on Genetic Engineering in 1974. During the 1980s, breakthroughs such as the production of

synthetic insulin, discovery of zinc finger nucleases (ZFNs), approval of the first recombinant

vaccine for humans, and Bt corn was first introduced in the market.

Further studies were performed during the 2000s. The first gene-targeted therapy was

sanctioned in 2001 while the pioneer FDA-approved cancer vaccine was released in 2006. In

addition to that, transcription activator-like effector nucleases (TALENs) was discovered in 2011

and CRISPR genome editing tools were discovered the year after. More recent studies since

2015 probed on genome editing using CRISPR and its subsequent approval for human trials in

2018.

Current Approaches and Applications

The four most common nuclease-based genome editing technologies utilize

meganucleases, ZFNs, TALENs, and CRISPR/Cas9. To target a precise section in the genome,

a double-strand break is first introduced via the programmable nucleases. The cell’s DNA repair

mechanism is then activated and the break is repaired either via nonhomologous end joining

(NHEJ) or homology-directed repair (HDR), resulting in cell lines with targeted gene

modifications, insertions, and deletions (Cox et al., 2015).

called meganucleases. They are characterized by their ability to recognize and excise large

portions of DNA sequences (Stoddard, 2006). Meganucleases can be divided into five

sub-families based on their sequence and structural motifs: LAGLIDADG, HNH, GIY-YIG,

His-Cys box, and PD-(D/E)XK. Among these meganucleases, the LAGLIDADG is the most

studied protein family because of its presence in all kingdoms of life and is generally encoded

within introns. As an RNA maturase, its main functions include facilitating the splicing of its own

intron. LAGDIDADG also works as a highly-specific endonuclease that recognizes and excises

the exon-exon junction sequence where the intron is located (Silva et al., 2011).

The next most common genome editing techniques are the chimeric enzymes ZFNs and

TALENs which are made up of a chimeric enzymes that contain a DNA-binding domain fused to

a bacterial restriction enzyme FokI nuclease (Ramirez et al., 2008) . ZFNs can trigger a highly

efficient gene targeting system and initiate precise double-strand breaks in various cell types

using an engineered zinc-finger array bound to a nuclease domain (Ramirez et al., 2008). At

present, ZFNs are frequently used in genetic engineering studies involving plant and animal

cells such as corn, soybean, rats, frogs, rabbits and many other mammalian cells. It is used in

creating isogenic human disease models, or cells that are designed to precisely model the

genome of a specific patient population (Torrance et al., 2001). These models are then used as

isogenic systems for high-throughput screening and drug discovery. For example, a study

conducted by Tebas et al. in 2014 revealed that human T-cells disrupted by ZFNs could

potentially be used as treatment for HIV/AIDS. Although it is relatively simple to predict

DNA-binding specific sites of zinc fingers, the problem arises when multiple zinc fingers are

needed to target unique sites in the human genome.

and are also currently used to recognize DNA-binding specific sites. In the Xanthomonas,

TALEs are injected into the plant cell via a type III secretion system that travels to the nucleus,

binds to the promoters of the target genes, and induces transcription (Boch, 2011). Different

TALEs recognize different binding sites because of the repeat variable dual residues which

specifies where the base is bound. As a result, each TALE can pinpoint a single base pair in a

continuous DNA sequence. Furthermore, this repeat domain rearrangement of TALE repeats is

interchangeable, resulting in novel DNA-binding specificities (Boch, 2011). This is also the

reason why some scientists suggest that TALEs with new specificities may be simpler to

manipule compared to zinc finger proteins (Boch, 2011).

Currently, TALENs have been used to genetically modify plants to create more

affordable food crops with better nutritional value (Zhang et al., 2013). Notable research studies

also include production of biofuels, engineering of stable modified human embryonic stem cells

and induced pluripotent stem cell clones and human erythroid cell lines (Hockemeyer et al.,

2011). Furthermore, use of TALENs have been explored experimentally correct

disease-causing genetic errors. For example, genetic defects that cause different diseases such

as sickle cell anemia and epidermolysis bullose have been experimented ​in vitro (Ramalingam

et al., 2014; Osborn et al., 2013). Recent studies, on the other hand, revealed that TALENs can

be used by the immune system since it can generate T-cells that show great anti-tumor potential

and are resistant to chemotherapeutic agents (Poirot et al., 2015).

The fourth major gene editing technique is the use of Clustered regularly interspaced

short palindromic repeats (CRISPR) and Cas9 (CRISPR-associated protein 9) protein system.
The CRISPR-Cas9 technology utilizes CRISPR sequences a guide to recognize and cleave

specific DNA strands that are complementary to its sequence (Zhang et al., 2014). This

DNA-encoded, RNA-mediated system provides precise genome targeting and degradation of

exogenous nucleic acid (Baranggou, 2015). Using this method, it is easier to target new DNA

sequences by changing only a small portion of the sequence of the corresponding RNA guide

with the matching base-pairs with the target DNA. In addition to that, a major advantage of Cas9

is its ability to induce multiplexing via expression of distinct guide RNAs, causing multiple

double-strand breaks in the same cell (Zhang et al., 2014). Application of CRISPR in studying

human development has also moved forward. Despite the popularity and success of human in

vitro fertilization, very little knowledge about the molecular stages of preimplantation,

implantation, and early placental formation in humans is known. These events are vulnerable to

disruption in both normal and IVF pregnancies since most of the knowledge comes from model

organisms. Although many of the major pathways are expected to be conserved, molecular and

morphological differences between mice and humans could significantly affect ​not only the

understanding of early pregnancy but also the production of pluripotent stem cells that are

suitable for use in developing stem cell-based therapies (Rossant and Tam, 2017).

From these, it can be seen that these four types of nucleases have been proven to be

effective gene editing techniques with a wide range of applications on model organisms and

mammalian cells. On-going studies in both academe and industry are being utilized to develop

these tools as therapeutics. However, some problems are still being encountered despite the

great advancements in this field.

Most of the problems encountered in genome editing involve risks for misplaced

double-strand breaks. Some guide RNAs don’t work as well and has the potential to either

disrupt the gene or cause chromosomal rearrangements once the break happens elsewhere in

the genome (Kohn et al., 2016). Furthermore, repeated repair of the double-strand break using

the NHEJ mechanism could lead to the formation of small insertion or deletion mutations

(indels) on the break site. Although many breakthroughs in the field of genome editing has been

coming into light, the danger from the effects of genotoxicity such as dysregulation,

trans-activation, and local gene inactivation should still be taken into consideration (Cox et al.,

2015).

Ethical Issues

Ethical issues over gene editing, especially when it comes to humans, have been an

issue ever since it was first discovered. Although gene editing refers to a general process, the

ethical implications regarding this technology are directly related to the purpose for which it is

intended to be used (Kohn et al., 2016). Presently, genome editing techniques have been

successfully used to alter genomes in individual cells and in non-human organisms. Genetic

modification has not only resulted in better targeted gene therapy in animal models of many

diseases but also herbicide and infection-resistant food crops (Kohn et al., 2016). However, the

potential applications that genome editing have are so broad and full of controversies, especially

when it involves alteration in the human germ-line. The possibility of manipulating the human

genome is very evident and while this could mean eventually eliminating dangerous genetic

conditions, it also poses a great risk to take advantage of eugenics.

reported. This prompted a series of responses from different organizations worldwide, calling for

a moratorium involving human embryo gene editing. As a result, the National Academy of

Sciences (NAS) and the National Academy of Medicine (NAM) spearheaded an international

summit in Washington last December 2015 to discuss the scientific, ethical, and societal issues

concerning human genome editing. A technical working group composed of experts like

research scientists, clinicians, and bioethicists from the field reviewed the current legal situation

and the extent of the ethical debate on genome editing. The committee’s peer-reviewed report

(National Academies of Sciences, Engineering, and Medicine et al., 2017) was released in

February 2017 and has been universally accepted as the guiding principles that can be used

across jurisdictions for applications in human gene editing. Major recommendations of the

committee are summarized in this report. The report did not condemn or ban germline gene

editing, however, they proposed an ironclad criteria and precautionary measures that need to be

considered before performing clinical trials. Furthermore, ​extension of human gene editing,

either somatic or germline, beyond the treatment or prevention of serious disease or for

enhancement purposes was clearly banned. Gene therapies which don’t cause any

non-inheritable changes in the human genome are ethically accepted while germline editing was

only allowed to treat serious genetic diseases where no reasonable alternative exists (Baltimore

et al., 2015).

The scientific community was shaken when He Jiankui, a Chinese Scientist, announced

in 2018 that he genetically modified human embryos. According to Jiankui, he made the twins

resistant to human immunodeficiency virus (HIV) by eradicating the CC5 gene from their genetic

make-up (Roberts, 2018). Many experts condemned his study, indicating that he clearly violated
the ethical guidelines for gene editing. They also cited that the effects of germ-line editing has

not been extensively studied yet, and that the alterations in the genome of the twins could

potentially harm the future generations who will inherit the changes (Roberts, 2018). Because of

this, the discussion about human germline editing continues and researches about gene therapy

are still regulated. Although the feasibility of correcting genes in human diploid zygotes (Ma et

al., 2017; Tang et al., 2017), and the discovery of new base editing tools to induce specific

nucleotide alterations are currently being explored in human embryos (Liang et al., 2017),

100% efficiency is still not attained and problems such as trans-activation, dysregulation, and

unwanted allelic variants hinder the success in clinical applications.

Following these ethical considerations, it is clear that there’s still a long way to go before

germline editing could be fully applied in human embryos, or if continuing this line of study will

be more beneficial than harmful to humans. Aside from that, authorities also face the challenge

of implementing and enforcing the proposed regulations and frameworks that are currently in

place. Nevertheless, one thing that can definitely be concluded from the present situation of

genome editing is that more research studies need to be conducted extensively to understand

the occurrences happening in the human genome. Transparency and initiating open discussions

about recombinant DNA technology is necessary to optimize the decisions about the future of

biology and genetics.

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