By:

omar abdelrahman ramadan

Biochemistry Pre-PhD - 2022

Gene editing and

gene therapy
Introduction
Genome editing (or Genome engineering) refers to altering an organism’s genetic code.
Alterations include deleting nucleotides to knock out a gene, adding nucleotides to knock in a
protein, or editing nucleotides to create a mutation. Gene editing can occur at the DNA, RNA,
or epigenetic level (Marchant, G.E 2021).

Genetic engineering of organisms is the key to biomedical and biotechnological research.

The ultimate tool would allow precise and unlimited modification of any given nucleic acid
sequence in a simple fashion. With the rise of genome editing, we have never been closer to
accomplishing this goal (Tröder and Zevnik 2022).

History of Genome editing

scientists have made incredible progress from the discovery of the structure of DNA to the
introduction of CRISPR. Here are some key discoveries in the history of genome engineering
that have brought us to where we are today.

Genetic engineering started with transgenesis, the deliberate transfer of genetic information
from one organism to another. In 1974, Rudolf Jaenisch demonstrated that viral DNA injected
into blastocysts led to stable integration in the genome of mice (Jaenisch, and Mintz, 1974). This is
considered to be the first transgenic animal generated. The term ‘transgenic’ though was introduced only
in 1981 by Gordon and Ruddle when they and others showed that injection of DNA into pronuclei of
murine zygotes conferred stable germline transmission of the genetic modification to the next generation
(Brinster et al., 1981)

• Discovery of the Double Helix (1953): DNA, as we know it today, was first
described as a double helix by James Watson, Francis Crick, and Rosalind Franklin
Klug, A. (2004).

• Creation of recombinant DNA (1972): Scientists were now able to artificially

introduce genetic code from one organism into another (Jaenisch, and Mintz, 1974).
• Generating transgenic animals (1981): The first transgenic animal, an animal
with foreign DNA inserted into its genome, was created when Thomas Wagner and
his team introduced a gene from a rabbit into a mouse (Brinster et al., 1981).
 • First genetically engineered drug (1982): Genentech scientists, Dennis Kleid and
David Goeddel, manufactured insulin in the laboratory, which led to approval for
human use by the FDA (Johnson, I. S, 2003).
• Discovery of Polymerase Chain Reaction - PCR (1983): PCR, a commonly used
technique to amplify a region of DNA, was first described by Kary Mullis (Bartlett
and Stirling, 2003).

• Sequencing the Human Genome (1999): The Human Genome Project, initiated in
the late 1990s, aimed to map out the entire human genome. Its completion in the
2000s has provided scientists with unparalleled knowledge of how certain diseases
and drug responses are linked to the human genome (Collins et al., 2003).
• CRISPR as a gene editing tool (2012): CRISPR was first described as a gene
editing tool, and its ability to specifically target, cut, and edit nucleotides has led to
transformative scientific discoveries ( Gasiunas et al., 2012).
• CAR-T therapy success (2017): Therapeutic applications of CRISPR were
highlighted with the success of CAR-T therapies for specific cancers in children
and adults, offering a potential alternative to chemotherapy in the future (Newick et
al ., 2017).

Gene Editing Techniques

Genetic modification techniques cover a wide range of studies, including the generation of
transgenic animals, functional analysis of genes, model development for diseases, or drug
development. The delivery of certain proteins such as monoclonal antibodies, enzymes, and
growth hormones has been suffering from several obstacles because of their large size. These
difficulties encouraged scientists to explore alternative approaches, leading to the progress in
gene editing. The distinguished efforts and enormous experimentation have now been able to
introduce methodologies that can change the genetic constitution of the living cell. The genome
editing strategies have evolved during the last three decades, and nowadays, four types of
“programmable” nucleases are available in this field: meganucleases, zinc finger nucleases,
transcription activator-like effector nucleases, and the clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system
(Khalil 2020).
• Meganucleases (MegNs):

Meganuclease technology involves re-engineering the DNA-binding specificity of

naturally occurring homing endo- nucleases. The largest class of homing
endonucleases is the LAGLIDADG family, which includes the well-characterized
and commonly used I- CreI and I-Sce I enzymes. Through a combination of rational
design and selection, these homing endo- nucleases can be reengineered to target
novel sequences. While many studies show promise for the use of meganucleases in
genome editing, the DNA-binding and cleavage domains of homing endonucleases
are difficult to separate, and the relative difficulty of engineering proteins with novel
specificities has traditionally limited the use of this platform. To address this
limitation, chimeric proteins comprising fusions of meganucleases, ZFs, and TALEs
have been engineered to generate novel monomeric enzymes that take advantage of
the binding affinity of ZFs and TALEs and the cleavage specificity of meganucleases.
One potential advantage associated with meganuclease technology is that DSB-
formation by these enzymes results in a 3’ overhang, which may be more
recombinogenic for HDR than the 5’ over- hang generated by FokI cleavage.
Additionally, meganucleases are the smallest class of engineered nucleases, making
them potentially amenable to all standard gene delivery methods. In fact, multiple
meganuclease monomers could be readily packaged into single viral vectors to
simultaneously create multiple DSBs (Maeder and Gersbach., 2016)

• Zinc finger nucleases:

Zinc finger (ZF) proteins are the most abundant class of transcription factors and the
Cys2-His2 zinc finger domain is one of the most common DNA-binding domains
encoded in the human genome. The crystal structure of Zif268 has served as the basis
for understanding DNA recognition by zinc fingers. In the presence of a zinc atom,
the zinc finger domain forms a compact ββα structure with the α-helical portion of
each finger making contact with 3 or 4 bp in the major groove of the DNA. Tandem
fingers in a zinc finger array wrap around the DNA to bind extended target sequences
such that a three-finger protein binds a 9 bp target site. The modular structure of
Zif268 suggested that these proteins might provide an attractive framework for
 engineering novel DNA-binding motifs. Initial attempts to design ZFs with unique
specificities based on a simple set of rules had some success however, combinatorial
libraries combined with selection-based
methods proved to be a more robust approach for generating individual fingers with
novel DNA-binding specificities. Following this success, the field was faced with the
challenge of engineering multi-finger arrays with novel target sites long enough to
be unique in a complex genome. The “modular assembly” approach relies on
collections of single-finger modules, either identified in naturally occurring proteins
or selected to bind specific three base pair target sites, which are then linked in
tandem to generate novel proteins. Alternatively, selection-based methods, such as
OPEN, may be used to select new proteins from randomized libraries. While
significantly more labor intensive, this method takes into account context-dependent
interactions between neighboring fingers within a multi-finger array. Several
methods, including those used by Sangamo Biosciences and the Sigma-Aldrich
CompoZr platform, combine these two approaches to assemble novel arrays using
archives of multi-finger units that have been preselected to work well together. The
zinc finger nuclease (ZFN) technology was made possible by the discovery that the

Figure 1 The principle of ZFN technique (Xiao et al., 2011) A ZFN monomer (two monomers are illustrated in the opposite direction
along the double strand DNA) is composed of a Fok I cleavage domain and a ZFP domain consisting of three zinc fingers. The ZFP domain
binds to one strand of a double-stranded DNA. The N-terminal of ZFP corresponds to the 3′-terminal of the target DNA. The −1, +3, +6
amino acid residue of each zinc finger recognizes three consecutive bases (a triplet) in the 3′→5′ direction to form a 9-bp recognition site.
The Fok I cleavage domain connects with the C-terminal of the ZFP; when the two Fok I domains in the opposite direction are spaced by
5–7 bp, they form a dimer and perform the cleavage function

DNA-binding domain and the cleavage domain of the FokI restriction endonuclease
function independently of each other. By replacing the FokI DNA-binding domain
 with a zinc finger domain, it is possible to generate chimeric nucleases with novel
binding specificities. Because the FokI nuclease functions as a dimer, two ZFNs
binding opposite strands of DNA are required for induction of a DSB. Initial
experiments showed that ZFN-induced DSBs could be used to modify the genome
through either NHEJ or HDR and this technology has subsequently been used to
successfully modify genes in human somatic and pluripotent stem cells (Maeder and
Gersbach., 2016).
• Transcription activator-like effector nucleases (TALE):

Transcription activator-like effector (TALE) nucleases (TALENs) have recently

emerged as a revolutionary genome editing tool in many different organisms and cell
types. The site-specific chromosomal double-strand breaks introduced by TALENs
significantly increase the efficiency of genomic modification. The modular nature of
the TALE central repeat domains enables researchers to tailor DNA recognition
specificity with ease and target essentially any desired DNA sequence. The scaffold
optimization isolated TALEN variants with high DNA cleavage efficiency, which is

Figure 2 The principle of TALEN technology (Mao and Tao 2015). a RVDs of TALE protein target and bind to dsDNA, and Fok I
cleaves dsDNA. b dsDNA breaks. c The broken dsDNA is repaired by NHEJ or HR

essential for targeted genome editing. The characterization of novel RVDs extended
the DNA recognition code and helped to minimize off-target cleavage activity of
TALENs by increasing guanine recognition specificity. Development of novel
strategies for convenient and quick assembly of TALE repeat arrays enabled high-
 throughput synthesis of TALENs and made TALEN technology accessible and
affordable for any academic or industrial lab (Sun and Zhao., 2013).

• Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) /

CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system
CRISPR/Cas9 is a third-generation gene editing technique that involves artificially
engineered immune system of bacteria and archaea, which mediates exogenous DNA
degradation and therefore serves as a defense mechanism against viral infections. The
clustered regularly interspaced short palindromic repeats (CRISPR) were first discovered in

Figure 3 The principle of the CRISPR/Cas9 system (Mao and Tao 2015). The transcribed pre-crRNA is processed by Cas 9 protein
and RNase III to a mature crRNA. The mature crRNA, Cas 9 and the transcribed tracrRNA bind together to form a complex. The complex
recognizes and opens the target DNA sequence which is complementary to the crRNA. The crRNA hybridizes with the complementary DNA
strand. Subsequently, the HNH activity site of the Cas9 protein cleaves the complementary DNA strand and the RuvC activity site cuts the
other free DNA strand to form a DNA double-strand break. When protospacer adjacent motifs (PAMs) are present, HR or NHEJ mechanism
is initiated to perform editing of the target gene
 the genome of E. coli in 1987, but did not arouse attention until 2012 when it was reported
that CRISPR and the CRISPR associated protein Cas9 cut DNA duplex at specific sites in vitro.
This finding potentiated the application of CRISPR/Cas9 in gene editing. 1 year later, two
CRISPR/Cas9 systems and demonstrated that Cas9 nucleases could be directed by short
RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells.
This is a breakthrough in gene editing research. CRISPR comprises a leader sequence,
several repeat sequences and several spacer sequences. The leader sequence is located
upstream of the CRISPR gene cluster. It has no coding activity, but serves as a species-
specific promoter. The repeat sequences are highly conserved palindromic sequences that
can form hairpin structures. The repeat sequences are not tandem arranged but
interrupted by spacer sequences. The spacer sequences are homologous to some
sequences in the genome of phages or plasmids, which enables the cells to recognize and
defend against the invasion of these corresponding phages or plasmids. Cas9 nuclease
complexes comprised of a Cas9 protein, a specific CRISPR RNA (crRNA), and a
transactivation CRISPR RNA (tracrRNA). The crRNA and tracrRNA form a dimer and bind to
the Cas9 protein. The complex recognizes and cuts target DNA at specific sites to generate
double-strand breaks that induce DNA repair in cells. In the absence of homologous DNA,
repair occurs through NHEJ, which facilitates gene deletion. When homologous DNA is
present, repair occurs through HR, which facilitates gene insertion (Jiang and Shen, 2019).

Gene Therapy:
Gene therapy is a medical approach that treats or prevents disease by correcting the
underlying genetic problem. Gene therapy techniques allow doctors to treat a disorder by altering
a person’s genetic makeup instead of using drugs or surgery. The earliest method of gene therapy,
often called gene transfer or gene addition, was developed to:

• Introduce a new gene into cells to help fight a disease.

• Introduce a non-faulty copy of a gene to stand in for the altered copy causing disease.

Later studies led to advances in gene therapy techniques. A newer technique, called genome editing
(an example of which is CRISPR-Cas9), uses a different approach to correct genetic differences.
Instead of introducing new genetic material into cells, genome editing introduces molecular tools
to change the existing DNA in the cell. Genome editing is being studied to:
 • Fix a genetic alteration underlying a disorder, so the gene can function properly.
• Turn on a gene to help fight a disease.
• Turn off a gene that is functioning improperly.
• Remove a piece of DNA that is impairing gene function and causing disease.

Gene therapies are being used to treat a small number of diseases, including an eye disorder called
Leber congenital amaurosis and a muscle disorder called spinal muscular atrophy. Many more
gene therapies are undergoing research to make sure that they will be safe and effective. Genome
editing is a promising technique also under study that doctors hope to use soon to treat disorders
in people.

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