Techniques in Molecular Biology and

Applications –

 Electrophoretic mobility-shift assay

 Chromatin immunoprecipitation
 CRISPR-Cas9
 RNA interference
 DNAse footprinting assay
Chromatin immunoprecipitation

• https://jove.com/v/5551/chromatin-immunoprecipitation
• https://www.cellsignal.com/applications/chip-and-chip-seq
WHAT IS A CHIP ASSAY?
• Chromatin immunoprecipitation (ChIP) assays are used to
evaluate transcription factor-DNA interactions and are
critical for advancing gene expression regulation and
epigenetic modifications studies. ChIP can detect and
relatively quantify specific protein-DNA and protein-protein
interactions in vivo at a single locus or multiple loci.

• ChIP involves chemically cross-linking proteins to DNA

sequences, which is followed by immunoprecipitation of the
cross-linked complexes (figure 1), and analysis of the
resultant DNA by endpoint or quantitative polymerase chain
reaction (qPCR) (figures 2-4), microarrays (ChIP-chip), or
next-generation sequencing (ChIP-seq) (figures 5 and 6).
• Protein and associated chromatin in living cells or tissues
are cross-linked using formaldehyde. The cross-linked DNA–
protein complexes (chromatin-protein) are then sheared into
∼500 bp DNA fragments using either enzymatic digestion or
physical shearing by sonication.
• The DNA-protein complexes are then immunoprecipitated
by an appropriate protein-specific antibody. After the cross-
links are reversed, the associated DNA fragments are eluted,
which is followed by immunoprecipitation of the cross-
linked complexes and analysis of the resultant DNA by
endpoint or quantitative polymerase chain reaction (qPCR),
microarrays (ChIP-chip), or next-generation sequencing
(ChIP-seq).
ChIP assays can be used for the following studies:
• DNA sequences occupied by multiple specific protein targets
• Binding sites and distribution of a particular protein, such as
a transcription factors, transcription co-factors, DNA
replication factors and DNA repair proteins throughout the
entire genome, under specified cellular conditions
• Gene transcription and polymerase activity
• Complex DNA/protein interactions underlying disease
phenotypes
• Modifications to proteins, such as histones, that may
influence chromatin structure and gene expression
• Nucleosome architecture and regulation of chromosomal
maintenance
• Chromatin immunoprecipitation (ChIP) is a type of
immunoprecipitation experimental technique used to
investigate the interaction between proteins and DNA in the
cell. It aims to determine whether specific proteins are
associated with specific genomic regions, such as
transcription factors on promoters or other DNA binding sites,
and possibly define cistromes.

• ChIP also aims to determine the specific location in the

genome that various histone modifications are associated
with, indicating the target of the histone modifiers. ChIP is
crucial for the advancements in the field of epigenomics and
learning more about epigenetic phenomena.
Briefly, the conventional method is as follows:
• DNA and associated proteins on chromatin in living cells or
tissues are crosslinked (this step is omitted in Native ChIP).
• The DNA-protein complexes (chromatin-protein) are then
sheared into ~500 bp DNA fragments by sonication or
nuclease digestion.
• Cross-linked DNA fragments associated with the protein(s)
of interest are selectively immunoprecipitated from the cell
debris using an appropriate protein-specific antibody.
• The associated DNA fragments are purified and their
sequence is determined. Enrichment of specific DNA
sequences represents regions on the genome that the
protein of interest is associated with in vivo.
Typical ChIP

• There are mainly two types of ChIP, primarily differing

in the starting chromatin preparation.
• The first uses reversibly cross-linked chromatin
sheared by sonication called cross-linked ChIP
(XChIP).
• Native ChIP (NChIP) uses native chromatin sheared
by micrococcal nuclease digestion.
Cross-linked ChIP (XChIP)
• Cross-linked ChIP is mainly suited for mapping the DNA
target of transcription factors or other chromatin-associated
proteins, and uses reversibly cross-linked chromatin as
starting material. The agent for reversible cross-linking could
be formaldehyde[3] or UV light
• Then the cross-linked chromatin is usually sheared by
sonication, providing fragments of 300 - 1000 base pairs (bp)
in length. Mild formaldehyde crosslinking followed by
nuclease digestion has been used to shear the chromatin.[5]
Chromatin fragments of 400 - 500bp have proven to be
suitable for ChIP assays as they cover two to three
nucleosomes.
• Cell debris in the sheared lysate is then cleared by sedimentation and
protein–DNA complexes are selectively immunoprecipitated using
specific antibodies to the protein(s) of interest. The antibodies are
commonly coupled to agarose, sepharose, or magnetic beads.
Alternatively, chromatin-antibody complexes can be selectively
retained and eluted by inert polymer discs.
• The immunoprecipitated complexes (i.e., the bead–antibody–protein–
target DNA sequence complex) are then collected and washed to
remove non-specifically bound chromatin, the protein–DNA cross-link
is reversed and proteins are removed by digestion with proteinase K.
An epitope-tagged version of the protein of interest, or in vivo
biotinylation can be used instead of antibodies to the native protein of
interest.
• The DNA associated with the complex is then purified and identified by
polymerase chain reaction (PCR), microarrays (ChIP-on-chip),
molecular cloning and sequencing, or direct high-throughput
sequencing (ChIP-Seq).[citati
Native ChIP (NChIP)

• Native ChIP is mainly suited for mapping the DNA target of histone
modifiers. Generally, native chromatin is used as starting chromatin.
As histones wrap around DNA to form nucleosomes, they are naturally
linked.
• Then the chromatin is sheared by micrococcal nuclease digestion,
which cuts DNA at the length of the linker, leaving nucleosomes intact
and providing DNA fragments of one nucleosome (200bp) to five
nucleosomes (1000bp) in length.
• Thereafter, methods similar to XChIP are used for clearing the cell
debris, immunoprecipitating the protein of interest, removing protein
from the immunoprecipitated complex, and purifying and analyzing
the complex-associated DNA.
Comparison of XChIP and NChIP
• The major advantage of NChIP is antibody specificity. It is important
to note that most antibodies to modified histones are raised against
unfixed, synthetic peptide antigens and that the epitopes they need
to recognize in the XChIP may be disrupted or destroyed by
formaldehyde cross-linking, particularly as the cross-links are likely
to involve lysine e-amino groups in the N-terminals, disrupting the
epitopes. This is likely to explain the consistently low efficiency of
XChIP protocols compared to NChIP.

• But XChIP and NChIP have different aims and advantages relative to
each other. XChIP is for mapping target sites of transcription factors
and other chromatin-associated proteins; NChIP is for mapping
target sites of histone modifiers (see Table 1).
• Comparison of ChIP-seq and ChIP-chip
• Chromatin Immunoprecipitation sequencing, also known as ChIP-seq, is an
experimental technique used to identify transcription factor binding events
throughout an entire genome. Knowing how the proteins in the human body
interact with DNA to regulate gene expression is a key component of our
knowledge of human diseases and biological processes. ChIP-seq is the
primary technique to complete this task, as it has proven to be extremely
effective in resolving how proteins and transcription factors influence
phenotypical mechanisms. Overall ChIP-seq has risen to be a very efficient
method for determining these factors, but there is a rivaling method known
as ChIP-on-chip.

• ChIP-on-chip, also known as ChIP-chip, is an experimental technique used to

isolate and identify genomic sites occupied by specific DNA-binding proteins
in living cells. ChIP-on-chip is a relatively newer technique, as it was
introduced in 2001 by Peggy Farnham and Michael Zhang. ChIP-on-chip gets
its name by combining the methods of Chromatin Immunoprecipitation and
DNA microarray, thus creating ChIP-on-chip.
• The two methods seek similar results, as they both strive to find
protein binding sites that can help identify elements in the human
genome. Those elements in the human genome are important for the
advancement of knowledge in human diseases and biological
processes.
• The difference between ChIP-seq and ChIP-chip is established by the
specific site of the protein binding identification. The main difference
comes from the efficacy of the two techniques, ChIP-seq produces
results with higher sensitivity and spatial resolution because of the
wide range of genomic coverage.
• Even though ChIP-seq has proven to be more efficient than ChIP-chip,
ChIP-seq is not always the first choice for scientists. The cost and
accessibility of ChIP-seq is a major disadvantage, which has led to the
more predominant use of ChIP-chip in laboratories across the world.
Table 1 Advantages and disadvantages of NChIP and XChIP

XChIP NChIP

Suitable for transcriptional factors, or any Testable antibody specificity

other weakly binding chromatin Better antibody specificity as target protein
Advantages associated proteins. naturally intact
Applicable to any organisms where native Better chromatin and protein revery
protein is hard to prepare efficiency due to better antibody specificity

Inefficient chromatin recovery due to

antibody target protein epitope disruption Usually not suitable for non-histone
May cause false positive result due to proteins
Disadvantages
fixation of transient proteins to chromatin Nucleosomes may rearrange during
Wide range of chromatin shearing size due digestion
to random cut by sonication.
• Limitations
• Large Scale assays using ChIP is challenging using intact
model organisms. This is because antibodies have to be
generated for each TF, or, alternatively, transgenic model
organisms expressing epitope-tagged TFs need to be
produced.
• Researchers studying differential gene expression patterns
in small organisms also face problems as genes expressed
at low levels, in a small number of cells, in narrow time
window.
• ChIP experiments cannot discriminate between different TF
isoforms (Protein isoform).
Electrophoretic mobility-shift assay
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757439/
 DNAse footprinting assay
• A DNase footprinting assay is a technique from molecular
biology/biochemistry that detects -DNA-protein interaction using the
fact that a protein bound to DNA will often protect that DNA from
enzymatic cleavage.

• This makes it possible to locate a protein binding site on a particular

DNA molecule. The method uses an enzyme, deoxyribonuclease
(DNase, for short), to cut the radioactively end-labeled DNA,
followed by gel electrophoresis to detect the resulting cleavage
pattern.
• For example, the DNA fragment of interest may be PCR
amplified using a 32P 5' labeled primer, with the result
being many DNA molecules with a radioactive label on
one end of one strand of each double stranded
molecule.

• Cleavage by DNase will produce fragments. The

fragments which are smaller with respect to the 32P-
labelled end will appear further on the gel than the
longer fragments. The gel is then used to expose a
special photographic film.
• The cleavage pattern of the DNA in the absence of a DNA binding
protein, typically referred to as free DNA, is compared to the
cleavage pattern of DNA in the presence of a DNA binding protein.
If the protein binds DNA, the binding site is protected from
enzymatic cleavage. This protection will result in a clear area on the
gel which is referred to as the "footprint".

• By varying the concentration of the DNA-binding protein, the

binding affinity of the protein can be estimated according to the
minimum concentration of protein at which a footprint is
observed.
Principles - DNase I footprinting assay is an in vitro method to
identify the specific site of DNA binding proteins. It not only finds
the target protein that binds to specific DNA, but also identify
which sequence the target protein is bound. This technique can be
used to study interactions between proteins and DNA both outside
and within cells.

• The protein binds to the DNA fragment, protecting the binding site
from cleavage by the DNase I. The fragments of the DNA
molecule are left after cleavage - also known as the
"footprinting", and thus its sequence can be determined. On the
autoradiogram of the polyacrylamide electrophoresis gel, there is
no radiolabeled band corresponding to the site of protein binding.
Footprinting assay is specific, accurate positioning, and widely
used.
• Regulation of transcription has been extensively
studied, but there is still much that is not known.
• Transcription factors and related proteins that
drive or repress transcription in conjunction with
promoters, enhancers, or silencers are crucial to
understanding the unique regulation of individual
genes within the genome.
• Techniques like DNase I footprinting help clarify
which proteins bind to these relevant regions of DNA
and reveal the complexities of transcriptional control.
Advanced Applications

1. In vivo footprinting - In vivo footprinting combined with immunoprecipitation can

be used to evaluate protein specificity at many positions throughout the genome.
DNA binding to the protein of interest can be immunoprecipitated with an
antibody to the protein, and then specific region binding can be assessed using
the DNA footprinting technique.

2. Quantitative footprinting - The DNA footprinting technique can be modified to

assess the binding strength of a protein to a DNA region. Using different
concentrations of the protein for the footprinting experiment, the appearance of
the footprint can be observed as the concentrations is increased and the
proteins binding affinity can then be estimated.

3. Detection by capillary electrophoresis - To adapt the footprinting technique to

newer detection methods, the labelled DNA fragments are detected by a
capillary electrophoresis apparatus rather than run on a polyacrylamide gel. If
the DNA fragment to be analyzed is generated by polymerase chain reaction
(PCR), a fluorescent molecule such as carboxyfluorescein (FAM) is directly
coupled to primers. This way, the fragments produced by DNase I digestion will
contain FAM, and may be detected by capillary electrophoresis machine.
RNA interference
https://en.wikipedia.org/wiki/RNA_interference

• RNA interference (RNAi) is a biological process in which RNA

molecules are involved in sequence-specific suppression of
gene expression by double-stranded RNA, through translational
or transcriptional repression.
• Historically, RNAi was known by other names, including co-
suppression, post-transcriptional gene silencing (PTGS), and
quelling.
• The detailed study of each of these seemingly different
processes elucidated that the identity of these phenomena
were all actually RNAi.
• Two types of small ribonucleic acid (RNA) molecules,
• microRNA (miRNA) and
• small interfering RNA (siRNA),
are central to components to the RNAi pathway.
• Once mRNA is degraded, post-transcriptional silencing occurs as
protein translation is prevented.

• Transcription can be inhibited via the pre-transcriptional silencing

mechanism of RNAi, through which an enzyme complex catalyzes
DNA methylation at genomic positions complementary to
complexed siRNA or miRNA.
• RNAi has an important role in defending cells against parasitic
nucleotide sequences (e.g., viruses or transposons) and also
influences development of organisms.
• MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that
help regulate gene expression, particularly during development.
• The phenomenon of RNAi, broadly defined, includes the endogenously
induced gene silencing effects of miRNAs as well as silencing triggered
by foreign dsRNA.
• Mature miRNAs are structurally similar to siRNAs produced from
exogenous dsRNA, but before reaching maturity, miRNAs must first
undergo extensive post-transcriptional modification.
• A miRNA is expressed from a much longer RNA-coding gene as a
primary transcript known as a pri-miRNA which is processed, in the cell
nucleus, to a 70-nucleotide stem-loop structure called a pre-miRNA by
the microprocessor complex.
• siRNAs derived from long dsRNA precursors differ from
miRNAs in that miRNAs, especially those in animals,
typically have incomplete base pairing to a target and inhibit
the translation of many different mRNAs with similar
sequences.
• In contrast, siRNAs typically base-pair perfectly and induce
mRNA cleavage only in a single, specific target.
• In Drosophila and C. elegans, miRNA and siRNA are
processed by distinct Argonaute proteins and Dicer
enzymes.
 RNA interference pathway
The term RNA interference (RNAi) was coined
to describe a cellular mechanism that uses
the gene's own DNA sequence to turn it off, a
process that researchers call silencing. In a
wide variety of organisms, including animals,
plants, and fungi, RNAi is triggered by
double-stranded RNA (dsRNA).

During RNAi, long dsRNA is cut or "diced"

into small fragments ~21 nucleotides long
by an enzyme called "Dicer". These small
fragments, referred to as small interfering
RNAs (siRNA), bind to proteins from a
special family: the Argonaute proteins.
After binding to an Argonaute protein, one
strand of the dsRNA is removed, leaving
the remaining strand available to bind to
messenger RNA target sequences
according to the rules of base pairing: A
binds U, G binds C, and vice versa. Once
bound, the Argonaute protein can either
cleave the messenger RNA, destroying it,
or recruit accessory factors to regulate the
target sequence in other ways.
• The RNAi pathway is a naturally occurring process found in
many eukaryotes and animal cells.
• It is initiated by the enzyme Dicer, which cleaves long double-
stranded RNA (dsRNA) molecules into short double-stranded
fragments of approximately 21 to 23 nucleotide siRNAs.
• Each siRNA is unwound into two single-stranded RNAs
(ssRNAs), the passenger (sense) strand and the guide
(antisense) strand.
• The passenger strand is then cleaved by the protein
Argonaute 2 (Ago2). The passenger strand is degraded and
the guide strand is incorporated into the RNA-induced
silencing complex (RISC).
• The RISC assembly then binds and degrades
the target mRNA.

• Specifically, this is accomplished when the

guide strand pairs with a complementary
sequence in a mRNA molecule and induces
cleavage by Ago2, a catalytic component of
the RISC. In some organisms, this process
spreads systemically, despite the initially
limited molar concentrations of siRNA.
• RNAi is a valuable research tool, both in cell culture and
in living organisms, because synthetic dsRNA
introduced into cells can selectively and robustly
induce suppression of specific genes of interest.
• RNAi may be used for large-scale screens that
systematically shut down each gene (and the
subsequent proteins it codes for) in the cell, which can
help to identify the components necessary for a
particular cellular process or an event such as cell
division. The pathway is also used as a practical tool
for food, medicine and insecticides.
Cellular mechanism
• The Dicer protein from Giardia
intestinalis, which catalyzes the
cleavage of dsRNA to siRNAs. The
RNase domains are colored green,
the PAZ domain yellow, the
platform domain red, and the
connector helix blue.
• RNAi is an RNA-dependent gene
silencing process that is
controlled by RISC and is initiated
by short double-stranded RNA
molecules in a cell's cytoplasm,
where they interact with the
catalytic RISC component
Argonaute. When the dsRNA is
exogenous (coming from infection
by a virus with an RNA genome or
laboratory manipulations), the
RNA is imported directly into the
cytoplasm and cleaved to short
fragments by Dicer. The initiating
dsRNA can also be endogenous
(originating in the cell), as in pre-
microRNAs expressed from RNA-
coding genes in the genome. The
primary transcripts from such
genes are first processed to form
the characteristic stem-loop
structure of pre-miRNA in the
nucleus, then exported to the
cytoplasm. Thus, the two dsRNA
pathways, exogenous and
endogenous, converge at the
RISC.
• Exogenous dsRNA initiates RNAi by activating the ribonuclease protein
Dicer which binds and cleaves dsRNAs in plants, or short hairpin RNAs
(shRNAs) in humans, to produce double-stranded fragments of 20–25
base pairs with a 2-nucleotide overhang at the 3′ end. Bioinformatics
studies on the genomes of multiple organisms suggest this length
maximizes target-gene specificity and minimizes non-specific
effects.

• These short double-stranded fragments are called siRNAs. These siRNAs

are then separated into single strands and integrated into an active RISC,
by RISC-Loading Complex (RLC).
• RLC includes Dicer-2 and R2D2 (effector proteins), and is crucial to unite
Ago2 and RISC. TATA-binding protein-associated factor 11 (TAF11)
assembles the RLC by facilitating Dcr-2-R2D2 tetramerization, which
increases the binding affinity to siRNA by 10-fold. Association with TAF11
would convert the R2-D2-Initiator (RDI) complex into the RLC.
• R2D2 carries tandem double-stranded RNA-binding domains to
recognize the thermodynamically stable terminus of siRNA duplexes,
whereas Dicer-2 the other less stable extremity. Loading is
asymmetric: the MID domain of Ago2 recognizes the
thermodynamically stable end of the siRNA. Therefore, the
"passenger" (sense) strand whose 5′ end is discarded by MID is
ejected, while the saved "guide" (antisense) strand cooperates with
AGO to form the RISC.

• After integration into the RISC, siRNAs base-pair to their target mRNA
and cleave it, thereby preventing it from being used as a translation
template. Differently from siRNA, a miRNA-loaded RISC complex
scans cytoplasmic mRNAs for potential complementarity. Instead of
destructive cleavage (by Ago2), miRNAs rather target the 3′
untranslated region (UTR) regions of mRNAs where they typically bind
with imperfect complementarity, thus blocking the access of
ribosomes for translation.
Three prime untranslated regions and microRNAs

• Three prime untranslated regions (3′UTRs) of mRNAs often contain

regulatory sequences that post-transcriptionally cause RNAi. Such 3′-
UTRs often contain both binding sites for miRNAs as well as for
regulatory proteins.
• By binding to specific sites within the 3′-UTR, miRNAs can decrease
gene expression of various mRNAs by either inhibiting translation or
directly causing degradation of the transcript. The 3′-UTR also may
have silencer regions that bind repressor proteins that inhibit the
expression of a mRNA.
• Crosstalk with RNA editing - The type of RNA editing that is most prevalent in higher eukaryotes
converts adenosine nucleotides into inosine in dsRNAs via the enzyme adenosine deaminase
(ADAR).[60] It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might
compete for a common dsRNA substrate.[61] Some pre-miRNAs do undergo A→I RNA editing[62][63]
and this mechanism may regulate the processing and expression of mature miRNAs.[63] Furthermore,
at least one mammalian ADAR can sequester siRNAs from RNAi pathway components.[64] Further
support for this model comes from studies on ADAR-null C. elegans strains indicating that A→I RNA
editing may counteract RNAi silencing of endogenous genes and transgenes.[65]
• Variation among organisms - Organisms vary in their ability to take up foreign dsRNA and use it in the
RNAi pathway. The effects of RNAi can be both systemic and heritable in plants and C. elegans,
although not in Drosophila or mammals. In plants, RNAi is thought to propagate by the transfer of
siRNAs between cells through plasmodesmata (channels in the cell walls that enable communication
and transport).[38] Heritability comes from methylation of promoters targeted by RNAi; the new
methylation pattern is copied in each new generation of the cell.[67] A broad general distinction
between plants and animals lies in the targeting of endogenously produced miRNAs; in plants,
miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce
direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and
induce translational repression.[66] This translational effect may be produced by inhibiting the
interactions of translation initiation factors with the mRNA's polyadenine tail.[68]
• Some eukaryotic protozoa such as Leishmania major and Trypanosoma cruzi lack the RNAi pathway
entirely.[69][70] Most or all of the components are also missing in some fungi, most notably the model
organism Saccharomyces cerevisiae.[71] The presence of RNAi in other budding yeast species such as
Saccharomyces castellii and Candida albicans, further demonstrates that inducing two RNAi-related
proteins from S. castellii facilitates RNAi in S. cerevisiae.[72] That certain ascomycetes and
basidiomycetes are missing RNAi pathways indicates that proteins required for RNA silencing have
been lost independently from many fungal lineages, possibly due to the evolution of a novel pathway
with similar function, or to the lack of selective advantage in certain niches.[73]
CRISPR-Cas9
What are genome editing and CRISPR-Cas9?

• Genome editing (also called gene editing) is a group of technologies that

give scientists the ability to change an organism's DNA.
• These technologies allow genetic material to be added, removed, or
altered at particular locations in the genome.
• Several approaches to genome editing have been developed. A well-
known one is called CRISPR-Cas9, which is short for clustered
regularly interspaced short palindromic repeats and CRISPR-
associated protein 9.
• The CRISPR-Cas9 system has generated a lot of excitement in the
scientific community because it is faster, cheaper, more accurate, and
more efficient than other genome editing methods.
• https://www.google.com/search?sca_esv=29f58c58bde83764&sc
a_upv=1&rlz=1C1CHBF_enIN1046IN1046&sxsrf=ACQVn0_KLGNJ
sxJ7Aid1jFmo5r6sll0_Fw:1713915867428&q=crispr+cas+techniq
ue&tbm=vid&source=lnms&prmd=ivnsbmtz&sa=X&ved=2ahUKE
wiHu8-
TwtmFAxXmzDgGHVVtA9MQ0pQJegQIERAB&biw=1280&bih=603
&dpr=1.5#fpstate=ive&vld=cid:44daa196,vid:MnYppmstxIs,st:0
• CRISPR (/ˈkrɪspər/)(an acronym for clustered regularly inters
paced short palindromic repeats) is a family
of DNA sequences found in
the genomes of prokaryotic organisms such
as bacteria and archaea.[2]
• These sequences are derived from DNA fragments
of bacteriophages that had previously infected the
prokaryote.
• They are used to detect and destroy DNA from similar
bacteriophages during subsequent infections. Hence these
sequences play a key role in the antiviral (i.e. anti-phage)
defense system of prokaryotes and provide a form
of acquired immunity.[2][3][4][5] CRISPR is found in
approximately 50% of sequenced bacterial genomes and
nearly 90% of sequenced archaea.[6]
• Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses
CRISPR sequences as a guide to recognize and open up specific
strands of DNA that are complementary to the CRISPR sequence.
• Cas9 enzymes together with CRISPR sequences form the basis of a
technology known as CRISPR-Cas9 that can be used to edit genes
within the organisms.[8][9]
• This editing process has a wide variety of applications including
basic biological research, development of biotechnological products,
and treatment of diseases.[10][11]
• The development of the CRISPR-Cas9 genome editing technique
was recognized by the Nobel Prize in Chemistry in 2020 which was
awarded to Emmanuelle Charpentier and Jennifer Doudna.[1
 Simplified diagram of a CRISPR locus. The
three major components of a CRISPR locus
are shown: cas genes, a leader sequence, and
a repeat-spacer array. Repeats are shown as
gray boxes and spacers are colored bars. The
arrangement of the three components is not
always as shown. In addition, several CRISPRs
with similar sequences can be present in a
single genome, only one of which is
associated with cas genes.

Mechanism of CRISPR-mediated immunity in

bacteria. 1. Adaptation insertion of new spacers
into the locus. 2. Expression processing of the
CRISPR RNA. 3. Interference detection of the
genetic elements by CRISPR RNA and Cas
proteins
• The Cas9 endonuclease is a four-component
system that includes two small molecules: crRNA
and trans-activating CRISPR RNA (tracrRNA).
• In 2012, Jennifer Doudna and Emmanuelle
Charpentier re-engineered the Cas9
endonuclease into a more manageable two-
component system by fusing the two RNA
molecules into a "single-guide RNA" that, when
combined with Cas9, could find and cut the DNA
target specified by the guide RNA
Q: How does the system work?
A: CRISPR “spacer” sequences are transcribed into short RNA
sequences (“CRISPR RNAs” or “crRNAs”) capable of guiding the system
to matching sequences of DNA.
When the target DNA is found, Cas9 – one of the enzymes produced by
the CRISPR system – binds to the DNA and cuts it, shutting the targeted
gene off.
Using modified versions of Cas9, researchers can activate gene
expression instead of cutting the DNA. These techniques allow
researchers to study the gene’s function.
• Research also suggests that CRISPR-Cas9 can be used to target and
modify “typos” in the three-billion-letter sequence of the human
genome in an effort to treat genetic disease.
•The CRISPR array is made up of an AT-rich
leader sequence followed by short repeats
that are separated by unique spacers.
• CRISPR repeats typically range in size from
28 to 37 base pairs (bps), though there can
be as few as 23 bp and as many as 55 bp.
Spacer acquisition
• When a microbe is invaded by a bacteriophage, the
first stage of the immune response is to capture
phage DNA and insert it into a CRISPR locus in the
form of a spacer. Cas1 and Cas2 are found in both
types of CRISPR-Cas immune systems, which
indicates that they are involved in spacer acquisition.
Mutation studies confirmed this hypothesis, showing
that removal of Cas1 or Cas2 stopped spacer
acquisition, without affecting CRISPR immune
response
Protospacer adjacent motifs (PAM)
• Bioinformatic analysis of regions of phage genomes that were
excised as spacers (termed protospacers) revealed that they were
not randomly selected but instead were found adjacent to short (3–5
bp) DNA sequences termed protospacer adjacent motifs (PAM).
• The PAM sequence appears to be important during spacer
insertion
• During the interference stage, the PAM sequence is recognized on
the crRNA-complementary strand and is required along with
crRNA annealing. Correct base pairing between the crRNA and the
protospacer signals a conformational change in Cascade that
recruits Cas3 for DNA degradation.
Mechanism of action of
CRISPR/Cas9
techniques.
CRIPSPR/Cas9 gene
with cleavage activities
creating a double-
strand break on the
target DNA, which are
repaired either by non-
homologous end
joining (NHEJ) or by
homology-directed
repair (HDR) with the
help of Donor DNA is
provided
• Working like genetic scissors, the Cas9 nuclease opens both
strands of the targeted sequence of DNA to introduce the
modification by one of two methods.
• Knock-in mutations, facilitated via homology directed repair
(HDR), is the traditional pathway of targeted genomic editing
approaches.
• This allows for the introduction of targeted DNA damage and
repair.
• HDR employs the use of similar DNA sequences to drive the
repair of the break via the incorporation of exogenous DNA to
function as the repair template.
• This method relies on the periodic and isolated occurrence of
DNA damage at the target site in order for the repair to
commence.
• Knock-out mutations caused by CRISPR-Cas9 result from the
repair of the double-stranded break by means of non-
homologous end joining (NHEJ) or POLQ/ polymerase theta-
mediated end-joining (TMEJ).
• These end-joining pathways can often result in random
deletions or insertions at the repair site, which may disrupt or
alter gene functionality.
• Therefore, genomic engineering by CRISPR-Cas9 gives
researchers the ability to generate targeted random gene
disruption.
• Because of this, the precision of genome editing is a great
concern.
• Genomic editing leads to irreversible changes to the genome.
• These differences may give Cas12a some advantages over Cas9. For
example, Cas12a's small crRNAs are ideal for multiplexed genome
editing, as more of them can be packaged in one vector than can
Cas9's sgRNAs.
• The sticky 5′ overhangs left by Cas12a can also be used for DNA
assembly that is much more target-specific than traditional restriction
enzyme cloning.
• Finally, Cas12a cleaves DNA 18–23 base pairs downstream from the PAM
site. This means there is no disruption to the recognition sequence after
repair, and so Cas12a enables multiple rounds of DNA cleavage.
• By contrast, since Cas9 cuts only 3 base pairs upstream of the PAM site,
the NHEJ pathway results in indel mutations that destroy the recognition
sequence, thereby preventing further rounds of cutting.
• Genome editing is of great interest in the prevention and treatment
of human diseases.
• Currently, genome editing is used in cells and animal models in
research labs to understand diseases.
• Scientists are still working to determine whether this approach is
safe and effective for use in people.
• It is being explored in research and clinical trials for a wide variety
of diseases, including single-gene disorders such as cystic
fibrosis, hemophilia, and sickle cell disease.
• It also holds promise for the treatment and prevention of more
complex diseases, such as cancer, heart disease, mental illness,
and human immunodeficiency virus (HIV) infection.
• While genome editing in eukaryotic cells has been possible using
various methods since the 1980s, the methods employed had proven to
be inefficient and impractical to implement on a large scale.
• With the discovery of CRISPR and specifically the Cas9 nuclease
molecule, efficient and highly selective editing became possible.
• Cas9 derived from the bacterial species Streptococcus pyogenes has
facilitated targeted genomic modification in eukaryotic cells by allowing
for a reliable method of creating a targeted break at a specific location
as designated by the crRNA and tracrRNA guide strands.
• The ease with which researchers can insert Cas9 and template RNA in
order to silence or cause point mutations at specific loci has proven to
be invaluable to the quick and efficient mapping of genomic models and
biological processes associated with various genes in a variety of
eukaryotes. Newly engineered variants of the Cas9 nuclease have been
developed that significantly reduce off-target activity.
• CRISPR-Cas9 genome editing techniques have many potential
applications.
• The use of the CRISPR-Cas9-gRNA complex for genome editing[10]
was the AAAS's choice for Breakthrough of the Year in 2015.[11] Many
bioethical concerns have been raised about the prospect of using
CRISPR for germline editing, especially in human embryos.[12]
• In 2023, the first drug making use of CRISPR gene editing, Casgevy,
was approved for use in the United Kingdom to cure sickle-cell
disease and beta thalassemia.[13][14] Casgevy was approved for
use in the United States on December 8, 2023, by the Food and
Drug Administration.[15]
• CRISPR-Cas systems have also been used for the
treatment of other hematologic diseases, such as
sickle cell disease (SCD) and hemophilia B (HB).
• SCD is a monogenic disease caused by a single-
nucleotide mutation in human β-globin gene, leading
to a substitution of glutamic acid by valine and the
production of an abnormal version of β-globin, which is
known as hemoglobin S (HbS).
• CRISPR-Cas9 system has been used to treat SCD by
repairing the β-globin gene mutation or reactivating
HbF expression
• HB is an X-linked hereditary bleeding disorder caused by deficiency of
coagulation factor IX, and the most common treatment for hemophilia B
is supplement blood coagulation factor
• Huai et al. injected naked Cas9-sgRNA plasmid and donor DNA into the
adult mice of F9 mutation HB mouse model for gene correction
• Meanwhile, Cas9/sgRNA were also microinjected into germline cells of
this HB mouse model for gene correction.
• Both in vivo and ex vivo experiment were sufficient to remit the
coagulation deficiency
• Guan et al. corrected the F9 Y371D mutation in HB mice using CRISPR-
Cas9 mediated in situ genome editing, which greatly improved the
hemostatic efficiency and increased the survival of HB mice
General applications of CRISPR
• Gene Therapy - The overall genetic disorders discovered till now are about
6000. Most of them do not have treatment till date. The role of gene therapy is
to substitute with exogenous DNA in the place of defective genes and edit the
mutated sequence. This therapy made a huge impact in medical
biotechnology.
• Base editing - They are two types of base editings: Cytidine base editor is a
novel therapy in which the cytidine (C) changes to thymidine (T). Adenine base
editor (ABE), in this there is a change in base complements from adenine (A) to
Guanine (G). The mutations were directly installed in cellular DNA so that
the donor template is not required. The base editings can only edit point
mutations moreover they can only fix up to four-point mutations. So, to master
this problem CRISPR system has introduced a new technique known as Cas9
fusion to stretch the level of genome editing.
• Gene silencing and activating -Furthermore, the CRISPR Cas9 protein can
modulate genes either by activating or silencing based on genes of interest.
There is a nuclease called dCas9 (endonuclease) used to silence or activate
the expression of genes.
Limitations in Applications of CRISPR
• The researchers are facing many challenges in gene editing.
• The major hurdles coming in the clinical applications are ethical issues
and the transport system to the target site. As the units of CRISPR
system taken from bacteria, when they are transferred to host cells it
produces an immune response against them.
• Physical, chemical, viral vectors are used as vehicles to deliver the
complex into the host. Due to this many complications are arising such
as cell damage that leads to cell death.
• In the case of viral vectors, the capacity of the virus is small and Cas9
protein is large. So, to overcome these new methods were developed in
which smaller strains of Cas9 are taken from bacteria. Finally, a great
extent of work is still needed to improve the system.
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