Genome Editing

Intro
Genome editing is a process that involves several actions affecting DNA
sequences in the genome, such as deletion, insertion or alteration of
these sequences. Researchers in the field of genome editing spent
many years attempting to create genome editing tools that were
effective, easy-to-use and non-expensive.

These tools could be used for a wide array of applications, such as

deleting the DNA sequences (gene) responsible for causing a particular
genetic disorder, which would vastly improve gene therapy in humans.
Another potential application would be for any industry that uses
bacteria to create product yields. The genomes of these bacteria could
be altered to optimize the amount of product they produce or the quality
of that product. Finally, a third example of a potential application would
be for agriculture. Using these tools to alter or manipulate a plant’s
genome could be used to lessen or eliminate plant diseases, and they
could be used to increase crop yields.

With all of these possibilities in the collective mind of the scientific

community, it was a huge breakthrough when CRISPR was developed.
CRISPR is a tool that operates on the molecular level and can edit DNA
sequences to a very specific and accurate degree. It can be
implemented to work on almost any locus. CRISPR has become
extremely popular and is starting to be used as a standard genome
editing tool in most research and development centres. CRISPR has
unlocked a lot of potential for a variety of applications.

Understanding CRISPR – The Basics

 Before CRISPR
Nowadays, CRISPR is ubiquitous in the world of gene editing, but to ZFNs, being composed of a DNA-binding domain and a second
it was not the first method of altering a genome. The earliest domain that cleaves the DNA. TALENs are more useful than ZFNs
methods came into use with the discovery of meganucleases and because their DNA-binding domains have more potential target
restriction enzymes. sequences, meaning they can be used for more applications.
TALENs were a step forward from ZFNs, but they were expensive to
The concept of accurate, specific genome editing was advanced produce, despite because easier to design than ZFNs.
by the development of zinc-finger nucleases (ZFNs) as a tool for
gene editing. A ZFN is an enzyme with both a zinc finger There is another gene editing technique that uses restriction
DNA-binding domain and also a restriction endonuclease domain. enzymes along with recombinant adeno-associated viruses
The first domain is used to target and bind to specific sequences (rAAVs). The adeno-associated virus is a non-pathogenic virus
of DNA and the second domain is used to cleave the DNA at the infects mammalian cells and affects their genomes at sites that can
target site. The zinc-finger domain is made of a 3-base pair be predicted. The virus genome can be altered to affect specific
segment of DNA that is designed to complement the target site target DNA. The main limitation with rAAVs is that their vectors have
and the restriction endonuclease cleaves the site that it is guided a very limited amount of genetic material to be altered, and therefore
to by the first domain. their resulting effectiveness is limited. Their vectors are also
difficult to produce.
ZFN was a huge breakthrough for site-specific gene editing, but
unfortunately it had some limitations. The results from ZFN use These gene editing tools all had limitations. ZFNs and TALENs were
showed off-target effects, ZFNs are difficult to engineer, making challenging to design and manipulate. rAAVs were limited by their
them expensive for researchers and time-consuming for small vector capacities. CRISPR, on the other hand, is based on
manufacturers. They were inefficient, and this meant that they DNA-RNA interactions, which are commonly understood, and
could only be applied to achieve one target change at a time. therefore, provides an easier, simpler way to edit genes. This
simplicity and effectiveness has changed the field of gene editing
Years later, transcription activator-like effector nucleases forever.
(TALENs) were developed. This method of gene editing is similar

Understanding CRISPR – The Basics

 CRISPR
CRISPR is made of two components which can be used to target the desired DNA sequence and then cleave the DNA at that
site. It can be used by researchers to alter a specific gene. Examples of the uses of CRISPR include knocking out a gene,
which means to make it inactive, or knocking in a gene, which could fix a deletion caused by mutation or add a beneficial trait
to the genome.

CRISPR – History
It began in 1993, when researchers identified repetitive The Cas9 molecule is guided to the target DNA by a guide RNA
palindromic segments of DNA in prokaryotes. These segments (gRNA). This short RNA fragment (the gRNA) is complementary to
were found among other sections of genetic material and were the target viral DNA sequence. This specific guiding system allows
soon called Clustered Regularly Interspaced Short Palindromic the Cas9 to cleave the DNA at a very specific level. This cleaving
Repeats (CRISPR). As it turned out, the sections of genetic process destroys the virus. Additionally, a piece of the foreign DNA
material between the CRISPR sections were actually the (referred to as a “spacer”) can be retained by the prokaryote and
interesting thing. This discovery built the foundation for the stored. The spacer will be kept in between the palindromic sequences
creation of CRISPR-Cas9 technology. of the CRISPR segments, which allows the prokaryote to retain a
memory of previous infections. In this way, any attempted re-infection
In 2007, a leap forward was made when researchers realised by the virus would be rapidly prevented and the attacking virus would
that the function of CRISPR was for prokaryotic immunity. The be destroyed. This is basically the equivalent of the human immune
underlying process, at a molecular level, was not fully system, which takes and retains antigens to prevent re-infection.
understood for another 5 years. Both bacteria and archaea (the
prokaryotes) use CRISPR-Cas9 to fight off invading viruses. Researchers quickly understood, after figuring out the CRISPR
When the viral infection occurs, the prokaryotic cell uses Cas9 mechanism in prokaryotes, that it could have massively beneficial
(which is a CRISPR-associated nuclease) to cleave the viral uses in humans, other animals, plants and microbes.
DNA by creating a double-strand break (DSB) in the target DNA
sequence.

Understanding CRISPR – The Basics

 CRISPR
Components
CRISPR has two components – a guide RNA (gRNA) and a
CRISPR-associated endonuclease (Cas). The gRNA is specific to the cas9

target DNA sequence. In experiments using CRISPR, the gRNA and

Cas are combined and the result is a ribonucleoprotein (RNP) sgRNA

complex.

Cas is a protein that acts to cut the target DNA while the gRNA guides Target DNA PAM
it to the target DNA site. In prokaryotes, the gRNA is used to target
viral DNA, but as a gene editing tool, it can be designed to target any
gene site in almost any location.

Cas9 is a popular choice of Cas proteins. Cas9 is from Streptococcus Figure 1.

pyogenes. The gRNA finds the target site and binds to the DNA, but
this binding requires the presence of a PAM (protospacer adjacent
motif) immediately downstream of the target but on the opposite DNA CRISPR-Cas9 is composed of a gRNA and Cas9 nuclease. Together,
these create a ribonucleoprotein (RNP) complex. A specific PAM
strand. Different Cas proteins (from different prokaryotic species)
must be present in the genomic DNA for the gRNA to be able to bind
recognise different PAM sequences. Cas9 is popular because of the to the target DNA segment. The Cas9 makes a double-strand break
frequency and flexibility of its PAM, which is 5’-NGG-3’. The N (DSB). Endogenous repair mechanisms react to the DSB and a
represents any nucleotide. This means that any DNA sequence with frameshift mutation may cause gene knockout, or they could cause
two G (guanine) bases can be used to form a PAM for Cas9. knock-in of a designated sequence. For knock-in, a DNA template
must be present.
If the gRNA successfully binds to the target, the Cas9 cleaves both
DNA strands. This cleaving process takes place 3 to 4 nucleotides
upstream of the PAM site.

Understanding CRISPR – The Basics

 Creating
Guide Double-Strand
RNA Breaks
In nature, gRNA is made of two separate sections of RNA. These Cas9 carries out its role in CRISPR-Cas gene editing experiments
two sections are CRISPR RNA (crRNA) and transactivating by causing a DSB to form in the DNA (Fig. 3). This resembles the
CRISPR RNA (tracrRNA). crRNA is 18-20 base pairs in length and process in prokaryotes. The DSB formation is the first step in the
binds to the DNA target sequence. TracrRNA acts as a structure CRISPR editing process. It is followed by a repair mechanism
which the crRNA-Cas9 interaction can take place on. In nature, which decides what type of gene editing will occur. Two types of
the gRNA is a duplex molecule, with crRNA and tracrRNA repair will be discussed next, namely non-homologous end
annealed together. Synthetically, gRNA can be produced with joining (NHEJ) and homology-directed repair (HDR).
these two molecules connected by a linker loop. These are called
single guide RNAs (sgRNAs).
linker loop

Target Target
RNA RNA
cas9
gRNA gRNA sgRNA

Target DNA PAM

DSB
gRNA Target Sequence gRNA Target Sequence

Figure 2. Figure 3.

Single guide RNAs and two component RNAs. The crRNA (green) and tracrRNA CRISPR-Cas9 produces a double strand break (DSB). Cas9 and sgRNA
(purple) components can be annealed together to form the two component combine to create a ribonucleoprotein. The sgRNA binds to the target DNA
gRNA, or the two components can be joined by a linker loop (blue) to create a and Cas9 cleaves the DNA, causing a DSB.
continuous molecule (sgRNA).

Understanding CRISPR – The Basics

 Double Strand Break Repair
As previously mentioned, CRISPR causes DSBs at desired sites in the genome. This is just the first step in the editing process. It is the
repair mechanisms that follow this that allow the editing to occur. Innate DNA repair processes automatically respond to the formation
of the DSB. The two main types of repair mechanisms that are used to edit genes are non-homologous end joining (NHEJ) and
homology-directed repair (HDR).

Non-Homologous End Joining DSB

(NHEJ)
Homology Arms

Knock-In Sequence

NHEJ can be used when the desired result is to DNA donar template

permanently knockout a gene (so that no functional

protein is made). (Fig. 4). NHEJ facilitates the re-joining of Non-Homologous End Homology-directed
Joining (NHEJ) Repair(HDR)
the DNA ends. However, it often allows erroneous changes
to occur, which may result in inserted or deleted
Insertion Knock-In Sequence
nucleotides that are not intended. These are called indels.
Or
If the number of nucleotide changes (inserted or deleted)
are not a multiple of three, a frameshift mutation will
occur. This mutation will likely eliminate the functionality
of the resulting protein. Deletion

Homology Directed Repair (HDR) Figure 4.

If the gene editing result desired by the researchers is to replace the targeted DNA sequence with another sequence, then HDR can be used.
A DNA template from a donor that possessed that desired DNA segment is introduced. This template is surrounded by sections of
homologous DNA sequences. The host’s repair mechanisms will use this template to fix the DSB by using homologous recombination. By
this process, the donor’s sequence is incorporated into the sequence being repaired.

Understanding CRISPR – The Basics

 Components of CRISPR-Cas9

3’ 5’
Linker
loop tracrRNA

cas9
3’
tracrRNA RNP

cas9 sgRNA 3’

or ~20 nt
gRNA
or
sgRNA
~20 nt gRNA
5’ 5’

gRNA Target Sequence

gRNA Target Sequence

3.Cas9-Mediated Double-Strand Break

NHEJ REPAIR
5’ 3’

3’ 5’

Insertion
5’ 3’
cas9 or
3’ 5’ 5’ 3’
sgRNA
DSB
3’ 5’
5’ 3’

3’ 5’ Deletion
Target DNA PAM

or

Homology-Directed Repair
Homology 5’ 3’
Arms
3’ 5’

Figure 5.
5’ 3’

3’ 5’

Knock-In Sequence
Knock-In Sequence
DNA Donor Template

Understanding CRISPR – The Basics

 What is
CRISPR Capable of?
The field of gene editing has grown vastly since CRISPR was developed. CRISPR can be used in a very large variety of cells in many
different organisms, such as mammals, plants and fungi. It has been used to alter genomes in many ways, including changes in
nucleotide sequences and changing the expression of genes. Figure 6 shows some of the current uses of CRISPR.

Gene tags Screening Anti-CRISPR CRISPRi/a Knock-ins Knockouts

Figure 6.

CRISPR is being used for many applications in gene editing, not just knocking in and knocking out genes. Research into CRISPR and
development of new CRISPR methods has led to several new applications of the technique. Examples include CRISPRi and CRISPRa,
anti-CRISPR proteins, CRISPR screens, and tagging genes via CRISPR to allow for tracking and visualization.

Understanding CRISPR – The Basics

 Knockouts
A knockout refers to the process of making a gene permanently
inactive, which means that it cannot encode a functioning protein.
CRISPR can cause this because of how error-prone NHEJ can be.
As previously described, NHEJ often produces indels that can
result in a frameshift mutation and inactivate the function of the
gene. A particularly susceptible DNA sequence is a premature
stop codon.
Hand photo created by teksomolika - www.freepik.com

Knockouts are also results of using multi-guide sgRNAs. These

are gRNAs that target the same gene. The subsequent multiple Knock-ins
cuts in the DNA induces at least one large fragment deletion in the
Knocking-in is the process of incorporating genetic material
target DNA. These deletions cause the loss of several amino
into a host genome. This process is facilitated by HDR, as
acids, which will likely result in the complete inactivation of the
discussed previously. In experiments using HDR, a DNA
target gene.
template is introduced to allow the HDR mechanism to
introduce the desired change into the host genome. HDR, as
The resulting changes in protein expression following a knockout a gene editing tool, allows for many applications. Examples
can provide researchers with an insight into the phenotype of a range from single point mutation alterations to the addition
cell or organism. This is useful for many applications, such as of selectable markers. The HDR mechanism still requires
identifying and validating potential drug targets, analysing further research to improve efficiency, but researchers have
cellular mechanisms and evaluating antibody activity. managed to use it to fix a mutation which causes cataracts
in mice. This illustrates that HDR is a viable concept for
correcting genetic diseases.

Understanding CRISPR – The Basics

 CRISPR
interference (CRISPRi) dCas9 Inhibitor
Gene Silenced

Knocking out is one way to disrupt gene expression by a particular

DNA sequence, but gene expression can also be suppressed in
another way that does not require the corresponding DNA to be
Promoter
altered. In 2013, Qi et al. developed a variant of Cas9 that does not
cleave DNA. This was achieved by altering the endonuclease
domains through mutation. This new variant is called dead Cas9 or
dCas9. Using dCas9, a new technique was created where the dCas9 dCas9 Activator Gene Expressed
binds to the target DNA (but does not cleave it) and prevents the host
cell’s transcription machinery from reaching the promoter. This
prevents the gene from being expressed without cleaving the DNA.
(Fig. 7a.) Combining a transcriptional repressor domain with dCas9 Promoter
produces a mechanism that is reversible and effective in inhibiting
gene expression.
Figure 7.
The term CRISPR interference is a reference to its precursor, the
gene-silencing technique RNA interference (RNAi). While RNAi CRISPR-Cas9 produces a double strand break (DSB). Cas9 and sgRNA
destroyed RNA transcripts to silence gene editing, CRISPRi affects combine to create a ribonucleoprotein. The sgRNA binds to the target DNA
and Cas9 cleaves the DNA, causing a DSB.
genes by impacting DNA. CRISPRi has higher efficiency, versatility
and less unintended (off-target) effects than RNAi.

CRISPR activation (CRISPRa)

dCas9 endonucleases can silence gene expression but they can also be used to activate desired genes
(called CRISPR activation). Combining a transcriptional activator with dCas9 can allow for editing that
results in target gene overexpression.
Understanding CRISPR – The Basics
 CRISPR
Screens
CRISPR can be used for functional screening of the genome. RNAi was the main method used for screening to test for gene loss of
function. This screening process involved the systematic inhibition of genes across the genome to ascertain their function. RNAi has
many problems, such as low efficiency and a high number of off-target effects. CRISPR is far more effective, with gRNA libraries that are
being developed to allow knocking out of hundreds of genes with high efficiency in one screening.

Anti-CRISPR
Cas9 + gRNA
CRISPR-Cas9 allows the fine-tuning of genomic DNA. CRISPR library Apply drug/
treatment
However, there is a downside. There is a risk of off-target Systematically disrupt a
effects, such as cleaving the DNA in the wrong location. set of genes using a
CRISPR library
The solution is to use anti-CRISPR proteins that inhibit the
activity of Cas9. This can be seen in nature, when
bacteriophages use these types of proteins to deflect the
prokaryote’s CRISPR machinery. This technology can be Figure 8.
applied to decrease errors in the editing process. When the gRNAs to target each gene are added to
ATGTCGTAGCGCCGTCGTAGC
CTCTCGTCGASGCAGSTGSCA
anti-CRISPR machinery is introduced after the editing each well of the well plate. A treatment, for GATCTGCAGCCCCGTCTATCT

process occurs, the cleavage at on-target sites is only example a drug, being applied to the target
cells would allow for the identification of
partially decreased, while cleavage at off-target sites is
which genes are responsible for the Identify cells with mutant
greatly decreased. increased or reduced sensitivity to the drug. phenotype and underlying
genotype

Understanding CRISPR – The Basics

 Gene
Visualizations
CRISPR can be used to visualize regions of the genome. This can be attained by the attachment of fluorescent proteins, such as green
fluorescent protein (GFP), to dCas9 or gRNAs, and then tagging the target areas of the genome with CRISPR. This allows researchers to
visualize nucleic acids, a process which was previously difficult to achieve in real time.

Steps
Design
An experiment with CRISPR begins with organising the parameters and
components for it. The gRNA must be designed and the suitable Cas nuclease
must be selected. Once they are completed, it is necessary to select the CRISPR
component format, choose a method of transfection, and prepare the optimal
conditions for the cell type being used. These steps will give a strong likelihood for
the success of the gene editing experiment.

Edit
After designing the experiment, you can begin to introduce the target-specific
gRNA and Cas9 nuclease to the cells. Carry out the transfection and allow CRISPR
to act.

Analyze
When the editing is completed, analyse the relevant genomic sequences to find
out the frequency and type of edits that have occurred in the genome. Several
analysis tools exist that can be used to evaluate the genomic results and Background photo created by kjpargeter - www.freepik.com

ascertain the editing efficiency. Protein and phenotypic assays are available to
evaluate gene editing effectiveness.
Understanding CRISPR – The Basics
 CRISPR
Future
CRISPR is extremely popular because of how specific and
feasible it is as a tool for genome editing. CRISPR has vastly
advanced and redefined the field of genome engineering.
CRISPR has facilitated many advances in recent years.
Researchers are attempting to develop the technology to
reveal its huge potential.

There is a modified version of CRISPR that is being used to

investigate epigenomics. This is a family of chemical groups
that are found throughout DNA and its associated histone
proteins. Previous to this CRISPR advancement, researchers
were limited to studying the correlation between gene
expression and epigenetic markers. This CRISPR complex can
acetylate histone proteins at desired locations. This can be Abstract photo created by kjpargeter - www.freepik.com
used to reveal more information about the causal relationship between gene expression and epigenetic markers.

CRISPR is becoming more important in the biomedical industry. Drug discovery and development is renowned as a long, expensive and
difficult process, but with CRISPR it is believed that the pre-clinical stage will become easier. An example is CRISPR screening libraries
which are now available to find new drug targets. Also, CRISPR can help to create accurate disease models for drug development, as well
as CRISPR research for in vivo and ex vivo therapies. Gene editing could greatly benefit the development of improved therapies and
medicines in the future.

CRISPR is advancing research into the most problematic areas of the field of biomedical science. It is also facilitating advances in other
fields of science, such as human therapeutics, agriculture, biofuels and general scientific research.

Understanding CRISPR – The Basics