ST A T E M ED I CA L A C A D EM Y

DNIPR OPET R OVSK

I N IC A L I M M U N O L OGY A N D
DEPART M EN T OF C L
OCCU P A T IO N A L D I SEA S E S
CRISPR TECHNOLOGY
-R.SUBASARAVANAN
CRISPR:
• CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic
Repeats. It is a family of DNA sequences in bacteria and archaea. The sequences
contain fragments of DNA from viruses that have attacked the prokaryote. These
fragments are used by the prokaryote to detect and destroy DNA from similar
viruses during subsequent attacks. These sequences play a key role in a
prokaryotic defense system, and form the basis of the CRISPR-Cas9 technology.
CRISPR SYSYEM:
• The CRISPR-Cas system is a prokaryotic immune system that confers resistance
to foreign genetic elements such as those present within plasmids and phages
that provides a form of acquired immunity . Upon the infection, new foreign DNA
sequences are captured and integrated into the host CRISPR locus as new
spacers. The CRISPR locus is transcribed and processed to generate
mature CRISPR RNAs (Cr RNAs), each encoding a unique spacer sequence.
Each crRNA associates with Cas effector proteins that use crRNAs as guides to
silence foreign genetic elements that match the crRNA sequence. Some Cas
(CRISPR-associated) proteins recognize and cut exogenous DNA. Other Cas
proteins cut foreign RNA . CRISPR systems are found in approximately 50% of
sequenced bacterial genomes and nearly 90% of sequenced archaea.
COMPONENTS OF CRISPR:
• Prokaryotes employ CRISPR-Cas (clustered regularly interspaced short
palindromic repeats and CRISPR-associated proteins) adaptive immune systems
to protect against viral infection. The CRISPR-Cas systems are encoded by
about 90% of archaea and 50% of bacteria. A typical CRISPR locus is
composed of an array of short direct repeats and interspersed spacer sequences
(short DNA sequences from invading viruses), which is flanked by diverse cas
genes. The rapid progression of CRISPR-Cas technology, from the discovery of
its DNA cutting-activity to its widespread adoption as a genome-editing tool, has
been astonishing. The technology have transformed scientists' ability to
manipulate genomes and study molecular networks, and concurrently opened
novel avenues for the treatment of genetic diseases.
• The CRISPR-Cas immunity response consists of three stages. During the
adaptation stage, also known as spacer acquisition, the processed foreign
DNA (known as the protospacer) is integrated into the host CRISPR locus,
yielding a new spacer. The CRISPR RNA (crRNA) expression and processing
stage involves transcription of the CRISPR locus into a single pre-crRNA and
further processing into mature crRNAs before being incorporated into the
CRISPR-Cas interference modules. In the interference stage, a single Cas
protein (or complex) uses the crRNA as a guide to cleave phage nucleic acid or
plasmid bearing a complementary sequence to the spacer sequence of the
crRNA. In type II CRISPR-Cas system, The sgRNA, which consists of crRNA-
and tracr-RNA-derived sequences, directs Cas 9 nuclease cleavage of the
corresponding target to initiate gene editing. All of the components of CRISPR
play important roles in the adaptive immune processes.
CRISPR MECHANISM:
 
• The key step in editing an organism's genome is selective targeting of a specific
sequence of DNA. Two biological macromolecules, the Cas9 protein and guide
RNA, interact to form a complex that can identify target sequences with high
selectivity. The Cas9 protein is responsible for locating and cleaving target DNA,
both in natural and in artificial CRISPR-Cas systems. The Cas9 protein has six
domains, REC I, REC II, Bridge Helix, PAM Interacting, HNH and RuvC.
• The Rec I domain is the largest and is responsible for binding guide RNA. The
role of the REC II domain is not yet well understood. The arginine-rich bridge
helix is crucial for initiating cleavage activity upon binding of target DNA .
The PAM-Interacting domain confers PAM specificity and is therefore
responsible for initiating binding to target DNA . The HNH and RuvC domains
are nuclease domains that cut single-stranded DNA. They are highly
homologous to HNH and RuvC domains found in other proteins  
• The Cas9 protein remains inactive in the absence of guide RNA . In
engineered CRISPR systems, guide RNA is comprised of a single strand of RNA
that forms a T-shape comprised of one tetraloop and two or three stem loops .
The guide RNA is engineered to have a 5′ end that is complimentary to the target
DNA sequence.
• Engineered guide RNA is a single strand of RNA. It forms one tetraloop and
two or three stem loops . This artificial guide RNA binds to the Cas9 protein and,
upon binding, induces a conformational change in the protein . The
conformational change converts the inactive protein into its active form. The
mechanism of the conformational change is not completely understood, but Jinek
and colleagues hypothesize that steric interactions or weak binding between
protein side chains and RNA bases may induce the change . 
• Once the Cas9 protein is activated, it stochastically searches for target DNA by
binding with sequences that match its protospacer adjacent motif (PAM)
sequence . A PAM is a two- or three-base sequence located within one
nucleotide downstream of the region complementary to the guide RNA. PAMs
have been identified in all CRISPR systems, and the specific nucleotides that
define PAMs are specific to the particular category of CRISPR system . The PAM
in Streptococcus pyogenes is 5′-NGG-3′.
• When the Cas9 protein finds a potential target sequence with the appropriate
PAM, the protein:guide RNA complex will melt the bases immediately upstream of
the PAM and pair them with the target complementary region on the guide RNA .
If the complementary region and the target region pair properly, the RuvC and
HNH nuclease domains will cut the target DNA after the third nucleotide base
upstream of the PAM.
ANTI-CRISPR:
• CRISPR-Cas systems are bacterial anti-viral systems. Prokaryotes utilize
CRISPR-mediated adaptive immune systems to kill the invading phages and
mobile genetic elements, and in turn, the viruses evolve diverse  anti- CRISPR
(Acr) proteins to evade that immunity. While Acrs are prevalent in phages
capable of lying dormant in a CRISPR-carrying host, their orthologs have been
observed only infrequently in virulent phages. The field of Acrs has rapidly
garnered interest, largely due to potential applications modulating the cleavage
activity of various Cas9 proteins. Tight control over Cas9 could prevent off-target
cleavage in genome-editing applications, or lock Cas9 into useful catalytically
inactive states. Bioinformatic methodologies have uncovered a number of Acrs
that interfere with different types of CRISPR-Cas systems in a variety of
manners.
• The proteins encoded by these genes are entirely distinct, with low sequence
similarity. Based on the classification of target CRISPR Immunity systems,
these proteins are divided into two classes, Class I anti-CRISPRs and Class II
anti-CRISPRs. Because many human pathogens encode CRISPR-Cas systems,
phages used for gene therapy could be outfitted with a variety of anti-CRISPRs
to expand their host range and prevent the bacterial adaptive immune response
from being mounted. The discovery of anti-CRISPR proteins has opened up a
new area of phage research and has provided a valuable addition to the CRISPR
toolbox. Although lots of anti-CRISPRs have been successfully identified, there is
a long way for us to go to unveil the details of this evolutionary war.
CRISPR – Cas9:
• The CRISPR-Cas9 system, which simply uses a guide RNA and Cas9 nuclease
to identify and cut target DNA sequences, comprises a robust technology that
has been used in diverse and innovative applications in biology. In the original
bacterial system, the chimeric CRISPR RNAs contain a "guide" sequence
homologous to the genome of the invading pathogen. The system works by Cas9
unfolding and running along the host DNA until reaching a region complementary
to the CRISPR guide sequence, which then binds to and cleaves the target site
by Watson-Crick base pairing and the endonuclease activity of the Cas9 protein.
This system has been adapted to introduce a genetic manipulation in cells by
designing CRISPR "guide" sequences from the organisms own genome. 
• The CRISPR-Cas9 system has incomparable advantages over other gene editing
tools. For example, the CRISPR-Cas9 system has more target sites than ZFNs
and TALENs, and Cas9 has many variants that can be used in a variety of
studies. Moreover, the system is extremely easy to use and can be executed in
almost any laboratory. CRISPR-Cas9-based tools have greatly enhanced our
ability to perform systematic analyses of gene function, as well as to reproduce
tumor-associated chromosomal translocations precisely. This technology has also
paved the way for the dissection of redundant gene functions, epigenetics and
possible gene therapy.
TYPES OF CRISPR - CAS SYSTEMS:
• The CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic
Repeats-CRISPR-associated protein system) is a bona fide adaptive (acquired)
immunity system that is present in most archaea and many bacteria. CRISPR-
Cas system is stored in the form of spacer sequences derived from foreign
genomes and inserted into CRISPR arrays, providing sequence-specific
protection against foreign DNA or, in some cases, RNA. 
• CRISPR-Cas is a programmable form of immunity that can adapt to target any
sequence and therefore is not under pressure to evolve an immense diversity of
specificities as is the case for restriction-modification enzymes, the most
abundant form of innate immunity in prokaryotes. Nevertheless, like any defense
system, CRISPR-Cas is engaged in an incessant arms race with viruses, which
results in rapid evolution of cas genes and notable diversity of the gene
repertoires and architectures of the CRISPR-cas loci translating into
diversification of the actual defense mechanisms. More specifically, the
diversification of the CRISPR-Cas systems is likely to be partly driven by their
competitive coevolution with virus-encoded dedicated anti-CRISPR proteins.
CLASSIFICATION OF CRISPR – CAS SYSTEMS:
• The CRISPR-Cas adaptive immune systems of prokaryotes split into two distinct
classes based on effector module organization. Class 1 CRISPR-Cas systems
utilize multi-protein effector complexes, whereas class 2 CRISPR-Cas
systems utilize single-protein effectors. Owing to the complexity of the gene
composition and genomic architecture of the CRISPR-Cas systems, any single,
all-encompassing classification criterion is rendered impractical, and thus a
'polythetic' approach based on combined evidence from phylogenetic,
comparative genomic and structural analysis was developed. Based on the two
distinct classes, the CRISPR-Cas system is further divided into six main types
(type I–type VI). Within each type of CRISPR-Cas system, several subtypes
have been delineated based on additional signature genes and characteristic
gene arrangements. 
TYPE I CRISPR - CAS SYSTEMS:
• Type I CRISPR-Cas systems exist in bacterial and archaeal organisms and
provide immunity against foreign DNA. The core defining feature of CRISPR-Cas
types and subtypes are the cas proteins, which are highly genetically and
functionally diverse, illustrating the many biochemical functions that they carry
throughout the processes of CRISPR-mediated immunity. Three major types of
the  CRISPR-Cas systems  distinguish from each other with the unique signature
genes: Cas3 in type I systems, Cas9 (formerly csn1) in type II, and Cas10 in
type III. In type I and type II systems, but not in type III, the selection of proto-
spacers in invading DNA depends on a proto-spacer-adjacent motif (PAM).
Moreover, related studies have revealed striking similarity in the organization of
effector complexes between type I and type III systems suggesting their origin
from a common ancestor.
IMPORTANT COMPONENTS IN TYPE I CRISPR-CAS SYSTEMS:
1. CAS3 PROTEIN: The signature gene, cas3, encodes a large protein with a
helicase possessing a single-stranded DNA (ssDNA)-stimulated ATPase activity
coupled to unwinding of double-stranded DNA (dsDNA) and RNA–DNA
duplexes. Often, but not always, the helicase domain is fused to an HD family
domain which has an endonuclease activity and is involved in the cleavage of
the target DNA. The HD domain is typically located at the N-terminus of Cas3
proteins (with the exception of subtype I-U and several subtype I-A systems, in
which the HD domain is at the carboxyl terminus of Cas3) or is encoded by a
separate gene within the same locus as cas3 helicase. In the latter case, the
helicase is denoted cas3′ and the HD nuclease is denoted cas3″. In type II and
type III systems, no Cas3 orthologue is involved.
2. CASCADES: The effector complexes of type I CRISPR-Cas display elaborate
architectures, with a backbone consisting of paralogous RAMPs (Repeat-
Associated Mysterious Proteins), such as Cas7 and Cas5, containing the
RRM (RNA Recognition Motif) fold and additional 'large' and 'small' subunits.
These effector complexes contain one Cas5 subunit and several Cas7 subunits.
The complex accommodates the guide RNA that consists of the spacer and a
portion of a repeat. The Cas5 subunit binds the 5′ handle of the crRNA and
interacts with the large subunit (Cas8). The small subunit is typically present in
several copies and interacts with the crRNA backbone bound to Cas7. Notably,
the length of the bound spacer correlates with the number of Cas7 subunits in
the backbone of the complex. An additional RAMP, Cas6, is loosely associated
with the effector complex and typically functions as the repeat-specific RNase in
the pre-crRNA processing.
MECHANISM OF TYPE I CRISPR-CAS SYSTEMS:
• CRISPR-based immunity contains three distinct stages: adaptation, expression,
and interference. Adaptation involves the acquisition of specific nucleotide
sequence tags, referred to as protospacers in their native context within invading
DNA, particularly bacteriophages and plasmids. During periods of predation,
protospacers are rapidly acquired and incorporated into the host genome, where
they are subsequently referred to as spacers. Cas1 and Cas2, which form a
complex that mediates acquisition of new spacers, are the only proteins
conserved between all CRISPR-Cas subtypes. Endonuclease activity of Cas1 is
required for spacer integration whereas Cas2 appears to perform a nonenzymatic
function. In type I and type II, protospacer-adjacent motif (PAM) palys important
roles in spacer acquisition and interference stages.
• In the second stage, CRISPR arrays are first transcribed into a single large pre-
crRNA by a promoter located within the CRISPR leader (lead), which is cleaved
into individual mature crRNAs by the Cas6 endonuclease (Type I and III
systems) or the ubiquitous RNase III enzyme (Type II systems). Processing
is mediated by characteristic secondary structures (hairpins) formed by Type I
pre-crRNAs or by a trans-activating RNA (tracrRNA) possessing homology to
direct repeat sequences in Type II systems. In type I, Cas6 is typically the
active endonuclease that is responsible for crRNA processing, and Cas5
and Cas7 are non-catalytic RNA-binding proteins; however, in type I-C
systems, crRNA processing is catalysed by Cas5. Once processed, crRNAs
form complexes with specific Cas proteins, including endonucleases responsible
for attack of invading DNA.
• In type I and type III CRISPR defence system, crRNA effector complexes
mediate the interference stage. In Type I systems, crRNAs complex with the
multiprotein 'Cascade' (comprised of cas6-cas8b-cas7-cas5 in Clostridium),
encoded downstream of the Type I CRISPR array, and base pair with invader
DNA, triggering nucleolytic attack by Cas3, whereas in type III-A and type III-B
systems the complexes are known as Csm and Cmr complexes, respectively. In
many CRISPR-Cas subtypes, Cascade may include Cas5, Cas6, Cas7, and
Cas8. The site of nucleolytic attack differs between CRISPR-Cas Types, as Cas
9 nuclease cleaves DNA 3 nucleotides upstream of the PAM resulting in a
double-stranded DNA break (DSB) in Type II systems, while Cas3 nicks the
PAM-complementary strand of invading DNA outside of the area of interaction
with crRNA, generating a DNA nick (DN). Nicked target DNA is subsequently
unwound and progressively degraded by Cas3.
TYPE Ⅱ CRISPR-CAS SYSTEMS:
• Type II CRISPR-Cas systems are considered to be the minimal CRISPR-Cas
systems that include the CRISPR repeat spacer array and only four (but
often three) cas genes, although additional bacterial factors, in particular trans-
activating crRNA (tracrRNA) and RNase III, contribute to the function of this
system. Similar to type I, type II CRISPR-Cas systems require a well-defined
short PAM that is located immediately downstream of the protospacer on the
non-target DNA strand. The PAM sequence is important both for spacer
acquisition and for target recognition and cleavage. Type II CRISPR systems are
the rarest, missing in archaea, and represented in ∼5% of bacterial genomes,
with an over-representation among pathogens and commensals. Recently, type
II systems have been developed into a powerful gene editing and engineering
tool with a major biotechnological potential.
APPLICATIONS TYPE Ⅱ CRISPR-CAS SYSTEMS:
• Type II CRISPR-Cas system has been adapted to be used in vitro, merging the
crRNA with a part of the tracrRNA in a hybrid called guide RNA (gRNA). Thus, a
variety of guide RNAs can be designed to direct the Cas9 endonuclease for site-
specific DNA cleavage and further genetic manipulations such as gene editing,
insertion or deletions. The easy conversion of Cas9 into a nickase was
utilized to facilitate homology directed repair in mammalian genomes with reduced
mutagenic activity and reported increased specificity. Furthermore, the DNA-
binding capacity of a catalytically inactive Cas9 mutant can be exploited to
engineer diverse RNA-programmable devices that can be used to mediate
transcriptional silencing or activation, or as DNA modification tools. The
unprecedented versatility in alternatives of genome engineering and modulation of
gene expression makes RNA-programmable Cas9 a unique technology in
molecular biology.
MECHANISM OF TYPE Ⅱ CRISPR-CAS SYSTEMS:
• Type II CRISPR-Cas systems incorporate sequences from invading nucleic
acid between CRISPR repeat sequences thanks to the Cas1-Cas2 complex.
The two proteins, that are present in the great majority of the known CRISPR-Cas
systems are sufficient for the insertion of spacers into the CRISPR arrays. The
endonuclease activity of Cas1 is required for spacer integration whereas
Cas2 appears to perform a nonenzymatic function. The Cas1-Cas2 complex
represents the highly conserved information processing module of CRISPR that
appears to be quasi-autonomous from the rest of the system.
• The minimal type II CRISPR-Cas systems employ an elaborate, unique
processing mechanism of pre-crRNA. Unlike most other systems of types I and III
that use a dedicated Cas endoribonuclease to cleave pre-crRNA by recognizing
the repeat units, type II systems use endogenous RNase III and a second
RNA called trans-activating CRISPR RNA (tracrRNA). The tracrRNAs have
been identified in most genomes encoding type II systems and are now
considered to be an integral component of this CRISPR type. The processing
event involves base-pairing of tracrRNA with the pre-crRNA repeats in the
presence of a nuclease and helicase protein, called Cas9, to form RNA duplexes
that are cleaved by the endogenous RNase III. The intermediate crRNAs undergo
further maturation resulting in mature individual crRNAs that remain duplexed
with tracrRNA in a complex with the Cas9 protein.
• Cas9 nuclease, the signature of type II CRISPR-Cas systems, is a large
multidomain protein that combines all the functions of effector complexes and the
target DNA cleavage and is essential for the crRNA maturation. Protospacer-
encoded portion of the crRNA directs Cas9 to cleave complementary target-DNA
sequences, if they are adjacent to short sequences known as protospacer
adjacent motifs (PAMs). PAMs are very important to the recognition of self and
non-self DNA because they are presents only in the foreign DNA sparing the
CRISPR mechanism to delete itself. Indeed, protospacer sequences incorporated
into the CRISPR locus are not cleaved presumably because they are not next to
a PAM sequence.
• 20 nucleotides at the 5' end of the gRNA direct Cas9 to a specific target DNA
site using standard RNA-DNA complementarity Watson-Crick base-pairing rules.
Cas9-induced DSBs are commonly repaired exploiting the NHEJ mediating indel
mutations as well as inducing HDR by providing single-stranded oligonucleotide
acting as a donor template. In the error-prone NHEJ pathway, the ends of DSB
are processed by endogenous DNA repair machinery and rejoined, which results
in random indel mutations at the site of junction which can result in the
frameshifts within the coding region of a gene and can cause the creation of a
premature stop codon, knocking out the target gene. Alternatively, when a repair
template in the form of a single-stranded oligodeoxynucleotide is supplied, the
HDR pathway allows high fidelity and precise editing.
TYPE Ⅲ CRISPR-CAS SYSTEMS:
• Type III CRISPR-Cas systems which are widespread in nature, are less useful as
a genetic tool but do also protect microbes from viruses. In the prokaryotic type
III systems, multiple Cas proteins assemble with crRNA into Csm (type III-
A/D) or Cmr (type III-B/C) effector complexes that provide interference against
invading nucleic acids through transcription-dependent DNA silencing. In addition
to targeting DNA, type III CRISPR targets the related RNA molecules from
viruses. When it encounters RNA from a virus, the type III CRISPR produces a
small molecule called cyclic oligoadenylate (cOA). The cOA molecule activates
Csm6 RNases known as non-specific ribonucleases, which can destroy all the
RNA in the cell. This defence is a less subtle than that provided type II CRISPR
and can also damage the cell by destroying other RNA molecules that the
microbes use to survive.
IMPORTANT COMPONENTS IN TYPE Ⅲ CRISPR-CAS SYSTEMS:
1. CSM/CMR COMPLEX: In type III systems, the interference complex (known
as Csm complex in type III-A/D systems and Cmr complex in type III-B/C
systems) is assembled from the signature multidomain protein Cas10 (Csm1
in type III-A/D or Cmr2 in type III-B/C systems) and additional Cas proteins
(Csm2-5 in type III-A/D, or Cmr1 and Cmr3-6 in type III-B/C). In these
systems, interference is transcription-dependent and is initiated by the crRNA-
guided interference complex binding to the nascent transcript of the target gene.
The target RNA-bound complex functions as a sequence-specific
endoribonuclease (RNase) to cleave the bound target RNA and in addition has a
target RNA-stimulated non-specific deoxyribonuclease (DNase) activity that
cleaves single-stranded DNA. Native Cmr complexes associate with multiple
size forms of crRNAs and cleave complementary target RNAs at multiple sites in
the region recognized by the crRNA.
• The type III-B Cmr immune effector complex is a multi-subunit noncoding RNP
that carries out RNA-guided cleavage of invader target RNAs in diverse bacterial
and archaeal organisms. crRNA binding requires four Cmr proteins. The 5' tag is
the defining feature of the crRNAs, is essential for Cmr complex formation and
function. Cmr3 cross-links with the signature 5' tag sequence of the crRNAs. The
interaction of Cmr3 with crRNAs also requires Cmr2, Cmr4, and Cmr5. Cmr2-5
form a complex with a crRNA interaction pattern and tag sequence dependence.
Each of the four proteins is required for formation of the complex. Remarkably,
efficient target RNA capture depends on the additional presence of Cmr1 and
Cmr6. Despite the presence of the crRNA, which includes a 37-nt guide region
that is complementary to the target RNA, the crRNP formed by Cmr2-5 does not
substantially interact with the target RNA.
2. CAS10 PROTEIN: The Cas10 subunit (called Csm1 and Cmr2 in the type III-
A/D and III-B/C systems, respectively) harbors an N-terminal HD domain, two
small a-helical domains, and two Palm domains that share a ferredoxin-like fold
with the core domain of nucleic acid polymerases and nucleotide cyclases. The
HD domain of Cas10 is responsible for ssDNA degradation in vitro. When crRNA
guides the Csm/Cmr complex to cleave the transcript, the single-stranded
deoxyribonuclease (ssDNase) activity of the Cas10 subunit is activated for
coupled degradation of the ssDNA in the transcription bubble. The conserved
GGDD motif in one of the two Palm domains can synthesise cyclic
oligoadenylate (cOA) molecules from ATP, when activated by target RNA binding.
cOA in turn binds to and activates Csm6 and Csx1, enhancing their ribonuclease
activity to degrade invader RNA transcripts, providing an additional interference
mechanism. cOA thus represents a new type of second messenger, generated
by type III CRISPR effector complexes, that sculpts the cellular response to
invasion by MGE. 
3. CSM6 PROTEIN: The CRISPR-associated protein Csm6 additionally contributes
to interference by functioning as a standalone ribonuclease that degrades
invader RNA transcripts. Members of the Csm6 or the related Csx1 protein
families are frequently encoded within type III CRISPR-Cas systems. The
proteins have an N-terminal CARF (CRISPR-associated Rossmann fold) domain
and a C-terminal HEPN (higher eukaryotes and prokaryotes nucleotide binding)
domain. Csm6 proteins function as RNases to non-specifically degrade
invader-derived RNA transcripts, providing an additional interference mechanism
that complements the nuclease activities of the type III interference complex.
Studies have shown that target RNA binding by the Csm effector complex of
Streptococcus thermophilus triggers Cas10 to synthesize cyclic oligoadenylates
by means of the Palm domains. Acting as signaling molecules, cyclic
oligoadenylates bind Csm6 to activate its nonspecific RNA degradation. This
cyclic oligoadenylate-based signaling pathway coordinates different components
of CRISPR-Cas to prevent phage infection and propagation.
MECHANISM OF TYPE Ⅲ CRISPR-CAS SYSTEMS:
• The interference complex of type III-A CRISPR-Cas systems is not only a
crRNA-guided RNase and DNase, but also a cyclic oligoadenylate
synthetase. Related studies have shown that the type III CRISPR interference
complex has three enzymatic activities: (i) a crRNA-guided endoribonuclease
activity against target RNA harboured by the Csm3 subunits, (ii) target RNA-
stimulated DNase activity harboured by the HD domain of Cas10 (Csm1 in
type III-A/D or Cmr2 in type III-B/C systems), (iii) target RNA-stimulated cyclic
oligoadenylate synthetase activity harboured by the Palm domain of Cas10.
• After infection, the transcription of phage DNA is initiated to establish and
maintain the infection cycle. In bacteria, the crRNA-guided Csm/Cmr complex
scans for the complementary target sequence in the invader's RNA. Tethering of
the Csm/Cmr complex to the transcript by crRNA triggers RNA cleavage by
Csm3/Cmr4 subunits and simultaneously activates the ssDNase activity of the
Cas10 subunit for coupled degradation of the ssDNA in the transcription bubble.
Csm/Cmr complexes avoid autoimmunity by checking the complementarity
between the crRNA 5′-handle, which originates from the CRISPR repeat, and the
3′-sequence flanking the target sequence in RNA. The cyclase domain of Cas10
can synthesise cOA from ATP, when activated by target RNA binding. cOA in turn
binds to and activates Csm6 and Csx1, enhancing their ribonuclease activity to
degrade invader RNA transcripts, providing an additional interference mechanism.
CRISPR IMMUNITY:
• Clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems
provide rapid and robust adaptation to the rapidly evolving viruses of archaea
and bacteria. Upon viral injection or plasmid invasion, a small DNA sequence
from invasive genetic elements, known as a spacer, is integrated into the
CRISPR loci to immunize the host cell. CRISPR loci consist of an array of short
and partially palindromic, repetitive sequences interspaced by equally short
'spacer' sequences. Spacers are transcribed into small RNA guides that direct
the cleavage of the viral DNA by Cas nucleases. Spacers are at the centre of
CRISPR defence as they specify immunity against phages or plasmids that
contain a complementary sequence. Immunization through spacer acquisition
enables a unique form of evolution whereby a population not only rapidly
acquires resistance to its predators but also passes this resistance
mechanism vertically to its progeny. 
• CRISPR-Cas immune systems function in three stages. The first stage is
spacer acquisition, also known as adaptation, in which new sequences from
exogenous nucleic acid are integrated into the CRISPR locus. The spacer
acquisition stage is followed by crRNA biogenesis, in which CRISPR loci are
transcribed and processed to generate small interfering CRISPR
RNAs (crRNAs) containing one spacer sequence. Spacer crRNA transcripts
provide the means for R-loop-based interference, when crRNA base pairs with
the complimentary DNA sequence in an mobile genetic element (MGE). The final
stage is targeting, in which the spacer sequences in these crRNAs are used
as guides to direct the specific cleavage of the viral genome by the Cas
nucleases. CRISPR – Cas systems have been classified into three major types,
namely Type I, Type II and Type III.
1. TYPE I CRISPR-CAS IMMUNITY:
• Genetically, Cas1 and Cas2 which are central to the immunization process of
universally occur across types and subtypes, whereas Cas3, Cas9 and Cas10
have been defined as the signature genes for Type I, Type II and Type III,
respectively. All types use the same basic molecular mechanism to achieve
immunity, that is, through crRNA-guided nucleases, while they differ in the
biogenesis of crRNAs and the targeting requirements.
• Type I CRISPR-Cas immunity is mediated by Cas3 nuclease and Cascade
complex. Cas6e, one of the subunits of Cascade complex, is a repeat-specific
endoribonuclease that cleaves the precursor crRNA which is generated by
transcription of the full CRISPR loci. The cleavage can generate short crRNAs
that remain associated with Cascade and that are used by the complex to locate
a complementary sequence in the target DNA, also known as the protospacer.
Another subunit, Cas8, also called as Cas A or Cse1, recognizes a short
sequence motif located immediately upstream of the target sequence
recognized by the crRNA. Sequence motifs adjacent to the targets specified by
CRISPR spacers were first identified in type II systems and subsequently named
as protospacer adjacent motif (PAM). PAM recognition is required for type I
CRISPR-Cas immunity, and the absence of a PAM in the repeat sequences
prevents the targeting of the spacers within the CRISPR loci by their
complementary crRNAs.
2. TYPE Ⅱ CRISPR-CAS IMMUNITY:
• Type Ⅱ CRISPR-cas systems  require only one cas gene, cas9, to execute
immunity in the presence of an existing targeting spacer sequence. However, as
opposed to the other CRISPR types, two small RNAs are needed for immunity:
the crRNA and the trans-encoded crRNA (tracrRNA). The tracrRNA forms a
secondary structure that mediates its association with Cas9 protein but also has a
region that is complementary to the repeat sequences of the CRISPR array. The
dsRNA formed between the tracrRNA and the precursor crRNA is cleaved by
RNase III, resulting in the cleavage of each repeat and the processing of the long
CRISPR transcript into small crRNA guides. Type II immunity also requires a PAM,
and mutations in this motif are the most common mechanism of escape from
CRISPR immunity by the targeted viruses.
• Type II CRISPR-Cas immunity results in the introduction of crRNA-specific dsDNA
breaks in the invading DNA that require two nuclease domains: HNH and RuvC.
Each of these domains cleaves one DNA strand of the protospacer sequence and
the tracrRNA is absolutely required for cleavage. The first step in target
recognition is the transient binding of Cas9 to PAM sequences within the
target DNA, which promotes the melting of the two DNA strands immediately
upstream of the PAM. A productive interaction in this region of the target, between
6-8 bases of the spacer sequence of the crRNA guide and the melted DNA,
triggers the formation of an R-loop and target cleavage.
3. TYPE Ⅲ CRISPR-CAS IMMUNITY
• In type III CRISPR-Cas systems, the precursor crRNA is cleaved by Cas6, a
repeat-specific endoribonuclease, which is not a subunit of the complex. As a
result of the cleavage, 8 nucleotides of the repeat sequence remain at the 5' end
of the spacer sequence in crRNA, a sequence known as the crRNA tag. Then the
resulting small crRNAs are transferred to a larger complex, the Cas10-Csm or
Cas10-Cmr complex for type III-A or III-B systems, respectively. Within these
complexes the crRNAs undergo a mature process whereby the 3' end is trimmed
at 6-nucleotide intervals. Type III CRISPR-Cas immunity requires target
transcription and a crRNA complementary to the non-template strand of the
DNA target and to the transcript. Both DNA and RNA are targeted by type III
CRISPR-Cas systems, resulting in the co-transcriptional crRNA-guided cleavage
of the target DNA and its transcripts. 
• The Cas10 complex contains both nucleolytic activities: the palm domain of
Cas10 is required for cleavage of the non-template DNA strand, and
backbone subunits Csm3 or Cmr4, for type III-A or III-B systems,
respectively, are responsible for cleavage of the RNA transcripts. To date, no
PAM requirements have been observed for type III CRISPR-Cas targeting. To
avoid targeting of the CRISPR locus, type III systems rely on the differential
base pairing between the crRNA tag and the sequences flanking the
protospacer. Whereas the absence of complete complementarity between these
sequences licenses DNA targeting, full complementarity between the crRNA tag
and the repeat sequence in the CRISPR locus prevents DNA targeting and thus
autoimmunity. 
CRISPR-CAS9 PROS AND CONS:
• The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-
associated protein (Cas)9 system has been rapidly developed, opening new
avenues for gene engineering. In recent years, CRISPR-Cas9 editing has been
implemented in a multitude of model organisms and cell types and has already
started to supplant incumbent genome editing technologies, such as TALENs
(transcription activator-like effector nucleases) and ZFNs (zinc finger nucleases).
CRISPR-Cas9 technology has evolved to create a simple, RNA-programmable
method to precisely mediate genome editing in mammalian cells. This
genome editing tool has improved our ability tremendously with respect to
exploring the pathogenesis of diseases and correcting disease mutations, as
well as phenotypes. Even if very efficient, this system is not completely immune
to errors, so understanding the possible weak sides could be helpful to prevent
all potential off-target effects.
ADVANTAGES OF CRISPR-CAS9 SYSTEM:
 The  CRISPR-Cas9 system  includes the RNA-guided Cas9 nuclease, which
binds to specific DNA sequences (complementary to the RNA-guide sequence)
and creates double-stranded breaks (DSB) on the DNA. The dsDNA breaks can
be repaired via homology-directed repair (HDR) or nonhomologous end-joining
(NHEJ). Based on this principle, the Cas9 and the guide-RNA were modified in
various ways to improve the efficiency and specificity of this system, to expand its
potential for different applications. This system can be used for altering specific
genetic loci through insertions, deletions, point mutations, and sequence
inversions. More recently, the system was modified to act as a genome regulator,
by tethering effector domains to the Cas9 or guide-RNA, and as a visualization
tool by fusing with marker molecules. This multiplex capacity of engineering
CRISPR-Cas9 enabled scientists to apply this system for genome modifications in
a variety of organisms.
 The CRISPR-Cas9 system comprises a robust technology that has been used in
diverse and innovative applications in biology. It has incomparable advantages
over other gene editing tools. For example, the CRISPR-Cas9 system has more
target sites than ZFNs and TALENs, and Cas9 has many variants that can be
used in a variety of studies. Moreover, the system is extremely easy to use and
can be executed in almost any laboratory. Cas9-based tools have greatly
enhanced our ability to perform systematic analyses of gene function, as well as
to reproduce tumor-associated chromosomal translocations precisely. This
technology has also paved the way for the dissection of redundant gene
functions, epigenetics and possible gene therapy. The CRISPR-Cas9 technology
is currently the simplest, most precise, and versatile method of genome
editing in a variety of cells and organisms, both in vitro and in vivo.
DISADVANTAGES OF CRISPR-CAS9 SYSTEM:
 To date, the CRISPR-Cas9 system has already shown itself to comprise a robust
and flexible tool for genome editing and gene regulation. With further research on
CRISPR, however, it became apparent that this technology was not as easy as
once assumed. Despite the many advantages of this system, there are some
challenges to the current Cas9-based tools. A large number of studies have
investigated diverse aspects that affect the efficiency and specificity of
CRISPR-Cas9 system, such as Cas9 activity, target site selection and
sgRNA design, delivery methods, off-target effects, and the incidence of
homology-directed repair (HDR). Many of the breakthroughs in the genome
engineering sphere have been viewed as huge scientific triumphs, but the
translation of these technologies from the bench to the bedside is not without
ethical, legal and social issues requiring vigorous debate.
 One potential limitation of CRISPR-Cas9 technology is that the approach may
create off-target effects. These off-target effects might play a role in
recognizing and destroying hypervariable viral nucleic acids or plasmid
DNA, which is beneficial to bacteria and archaea. However, for biological
studies and genetic therapies, off-target phenomena generate undesired
mutations at random sites, thus impacting precise gene modification. We
must be wary of the potential consequences of off-target effects, lack of specificity
in targeting, incomplete targeting, and so on, all of which could have devastating
effects on patients. Scientists will be looking for improved nucleases to increase
safety and efficacy. In particular, the improved nucleases hold promise for
reducing off-target effects, which would improve the effectiveness of the target
gene edit, and reduce unintended consequences. Greater accuracy will also
improve our ability to characterize mechanism and to monitor efficacy and safety,
including over multiple generations.
 Ethical concerns about germline gene editing is also a contentious issue.
There are myriad concerns about the introduction of heritable modifications
should any of the manipulated embryos be used in reproduction. There are
concerns about the risk of errors and unintended consequences, not only for the
resulting children but also for humankind. In the latter context there are concerns
about the risk of exacerbating problems of racism, sexism, health inequality, and
so on, as a direct consequence of who will and who will not have access to the
technology. A bigger obstacle to the emergence of "designer babies" and
Gattaca-type dystopian futures: the principles of evolution. Gene editing
could ruin human evolution. Though some challenges remain ahead, the
application of this technology to several aspects of sarcoma biology, ranging from
basic research to clinical and translational applications, offers numerous exciting
opportunities for a better understanding and potential treatment of these
devastating diseases.