CRISPR

CRISPR (an acronym for clustered regularly interspaced short palindromic

repeats) is a family of DNA sequences found in
the genomes of prokaryotic organisms such as bacteria and archaea.

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.
CRISPR is found in approximately 50% of sequenced bacterial
genomes and nearly 90% of sequenced archaea.

CRISPR Cascade protein (cyan) bound to CRISPR RNA (green) and phage DNA (red)

Cas-9 (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.
This editing process has a wide variety of applications including basic biological
research, development of biotechnological products, and treatment of diseases.

 The development of the CRISPR-Cas9 genome editing technique was

recognized by the Nobel Prize Chemistry in 2020 which was awarded
to Emmanuelle Charpentier and Jennifer Doudna.
HISTORY
Repeated sequences

The discovery of clustered DNA repeats took place independently in three parts of
the world. The first description of what would later be called CRISPR is from Osaka
University researcher Yoshizumi Ishino and his colleagues in 1987.

They accidentally cloned part of a CRISPR sequence together with the "iap"
gene (isozyme conversion of alkaline phosphatase) from the genome
of Escherichia coli which was their target. The organization of the repeats was
unusual. Repeated sequences are typically arranged consecutively, without
interspersing different sequences. They did not know the function of the
interrupted clustered repeats.

 In 1993, researchers of Mycobacterium tuberculosis in the Netherlands

published two articles about a cluster of interrupted direct repeats (DR) in
that bacterium. They recognized the diversity of the sequences that
intervened in the direct repeats among different strains of M. tuberculosis
and used this property to design a typing method that was named
spoligotyping (A technique for the identification and analysis of
polymorphism in certain types of repeats unit in DNA), which is still in use,
today.

Francisco Mojica at the University of Alicante in Spain studied repeats observed in

the archaeal organisms of Haloferax and Haloarcula species and their function.
Mojica's supervisor surmised at the time that the clustered repeats had a role in
correctly segregating replicated DNA into daughter cells during cell division
because plasmids and chromosomes with identical repeat arrays could not coexist
in Haloferax volcanii. Transcription of the interrupted repeats was also noted for
the first time; this was the first full characterization of CRISPR.

 By 2000, Mojica performed a survey of scientific literature and one of his

students performed a search in published genomes with a program devised
 by himself. They identified interrupted repeats in 20 species of microbes as
belonging to the same family
Because those sequences were interspaced, Mojica initially called these
sequences "short regularly spaced repeats" (SRSR).

 In 2001, Mojica and Ruud Jansen, who were searching for a additional
interrupted repeats, proposed the acronym CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats) to alleviate the confusion
stemming from the numerous acronyms used to describe the sequences in
the scientific literature.

 In 2002, Tang, et al. showed evidence that CRISPR repeat regions from the
genome of Archaeoglobus fulgidus were transcribed into the long RNA
molecules that were subsequently processed into unit-length small RNAs,
plus some longer forms of 2, 3, or more spacer-repeat units.

Timeline of the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)–Cas system with
important milestones
CRISPR-associated systems
A major addition to the understanding of CRISPR came with Jansen's observation
that the prokaryote repeat cluster was accompanied by a set of homologous
genes that make up CRISPR-associated systems or cas genes. Four cas genes
(cas 1–4) were initially recognized. The Cas proteins
showed helicase and nuclease motifs, suggesting a role in the dynamic structure
of the CRISPR loci .
In this publication, the acronym CRISPR was used as the universal name of this
pattern. However, the CRISPR function remained enigmatic.

In 2005, three independent research groups showed that some CRISPR spacers
are derived from phage DNA and extrachromosomal DNA such as plasmids.
In effect, the spacers are fragments of DNA gathered from viruses that previously
tried to attack the cell. The source of the spacers was a sign that the CRISPR-
cas system could have a role in adaptive immunity in bacteria.All three studies
proposing this idea were initially rejected by high-profile journals, but eventually
appeared in other journals

The first publication proposing a role of CRISPR-Cas in microbial immunity, by

Mojica and collaborators at the University of Alicante,predicted a role for the RNA
transcript of spacers on target recognition in a mechanism that could be
analogous to the RNA interference system used by eukaryotic cells. Koonin and
colleagues extended this RNA interference hypothesis by proposing mechanisms
of action for the different CRISPR-Cas subtypes according to the predicted
function of their proteins.

Experimental work by several groups revealed the basic mechanisms of CRISPR-

Cas immunity.

 In 2007, the first experimental evidence that CRISPR was an adaptive

immune system was published. A CRISPR region in Streptococcus
thermophilus acquired spacers from the DNA of an infecting bacteriophage.
The researchers manipulated the resistance of S. thermophilus to different
types of phages by adding and deleting spacers whose sequence matched
those found in the tested phages.
  In 2008, Brouns and Van der Oost identified a complex of Cas proteins
(called Cascade) that in E. coli cut the CRISPR RNA precursor within the
repeats into mature spacer-containing RNA molecules called CRISPR RNA
(crRNA), which remained bound to the protein complex.

Moreover, it was found that Cascade, crRNA and a helicase/nuclease (Cas3) were
required to provide a bacterial host with immunity against infection by a DNA
Virus. By designing an anti-virus CRISPR, they demonstrated that two orientations
of the crRNA (sense/antisense) provided immunity, indicating that the crRNA
guides were targeting dsDNA.
That year Marraffini and Sontheimer confirmed that a CRISPR sequence
of S.epidermis targeted DNA and not RNA to prevent conjugation. This finding
was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas
immunity, although a CRISPR-Cas system that targets foreign RNA was later found
in Pyrococcus furiosus.

 A 2010 study showed that CRISPR-Cas cuts both strands of phage and
plasmid DNA in S. thermophilus.
Cas9
A simpler CRISPR system from Streptococcus pyogenes relies on the
protein Cas9. The Cas9 endonulclease is a four-component system that
includes two small molecules: crRNA and trans-activating CRISPR RNA
(tracrRNA).

 In 2012, Jennifer Doudna and Emannuelle 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.This contribution was so
significant that it was recognized by the Nobel Price in Chemistry in
2020.
 By manipulating the nucleotide sequence of the guide RNA, the
artificial Cas9 system could be programmed to target any DNA
sequence for separation.
Another group of collaborators comprising Virginijus Siksyns together
with Gasiūnas, Barrangou, and Horvath showed that Cas9 from the S.
thermophilus CRISPR system can also be reprogrammed to target a site
of their choosing by changing the sequence of its crRNA. These
advances fueled efforts to edit genomes with the modified CRISPR-
Cas9 system.

 Groups led by Feng Zhang and George Church simultaneously

published descriptions of genome editing in human cell cultures
using CRISPR-Cas9 for the first time.It has since been used in a wide
range of organisms, including baker's yeast (Saccharomyces
Cerevisiae),the opportunistic pathogen Candida albicans,zebrafish
(Danio rerio),fruit flies (Drosophila melanogaster),ants
(Harpegnathos saltator and Ooceraea biroi ), mosquitoes ( Aedes
aegypti ) ,nematodes (Caenorhabditis elegans),plants ,mice (Mus
musculus domesticus),monkeys and human embryos.

CRISPR has been modified to make programmabletranscriptio factor that

allows targeting and activation or silencing specific genes.

 The CRISPR-Cas9 system has shown to make effective gene edits in

Human tripronuclear zygotes first described in a 2015 paper by
Chinese scientists P. Liang and Y. Xu. The system made a successful
cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos.
4 out of the 28 embryos were successfully recombined using a donor
template given by the scientists. The scientists showed that during
DNA recombination of the cleaved strand, the homologous
endogenous sequence HBD competes with the exogenous donor
template. DNA repair in human embryos is much more complicated
and particular than in derived stem cells.

Cas12a
In 2015, the nuclease Cas12a (formerly known as Cpf1) was characterized in
the CRISPR-Cpf1 system of the bacterium Francisella novicida. Its original name,
from a TIGRFAMs protein family definition built in 2012, reflects the prevalence of
its CRISPR-Cas subtype in the Prevotella and Francisella lineages.
Cas12a showed several key differences from Cas9 including: causing a 'staggered'
cut in double stranded DNA as opposed to the 'blunt' cut produced by Cas9,
relying on a 'T rich' PAM (providing alternative targeting sites to Cas9), and
requiring only a CRISPR RNA (crRNA) for successful targeting. By contrast, Cas9
requires both crRNA and a transactivating crRNA (tracrRNA).

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.
In theory, repeated rounds of DNA cleavage should cause an increased
opportunity for the desired genomic editing to occur.

A distinctive feature of Cas12a, as compared to Cas9, is that after cutting its

target, Cas12a remains bound to the target and then cleaves other ssDNA
molecules non-discriminately.This property is called "collateral cleavage" or
"trans-cleavage" activity and has been exploited for the development of various
diagnostic technologies
A diagram of the CRISPR nucleases Cas12a and Cas9 with the position of DNA cleavage

Cas13

In 2016, the nuclease Cas13a (formerly known as C2c2) from the

bacterium Leptotrichia shahii was characterized. Cas13 is an RNA-guided
RNA endonuclease, which means that it does not cleave DNA, but only
single-stranded RNA. Cas13 is guided by its crRNA to a ssRNA target and
binds and cleaves the target. Similar to Cas12a, the Cas13 remains bound
to the target and then cleaves other ssRNA molecules non-
discriminately. This collateral cleavage property has been exploited for the
development of various diagnostic technologies.

In 2021, Dr. Hui Yang characterized novel miniature Cas13 protein

(mCas13) variants, Cas13X & Cas13Y. Using a small portion of N gene
sequence from SARS-CoV-2 as a target in characterization of mCas13,
revealed the sensitivity and specificity of mCas13 coupled with RT-LAMP
for detection of SARS-CoV-2 in both synthetic and clinical samples over
other available standard tests like RT-qPCR .
Locus structure

Repeats and spacers

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. Some show dyad symmetry,
implying the formation of a secondary structure such as a stem-loop ('hairpin') in
the RNA, while others are designed to be unstructured. The size of spacers in
different CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp). New spacers
can appear rapidly as part of the immune response to phage infection.There are
usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array.

CRISPR-DR2: Secondary structure CRISPR-DR5:Secondary structure

CRISPR-DR8: Secondary structure CRISPR-DR9: Secondary structure

Cas genes and CRISPR subtypes

Small clusters of cas genes are often located next to CRISPR repeat-spacer arrays.
Collectively the 93 cas genes are grouped into 35 families based on sequence
similarity of the encoded proteins. 11 of the 35 families form the cas core, which
includes the protein families Cas1 through Cas9. A complete CRISPR-Cas locus has
at least one gene belonging to the cas core.
CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of
multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single
large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV;
class 2 is divided into types II, V, and VI. The 6 system types are divided into 19
subtypes. Each type and most subtypes are characterized by a "signature gene"
found almost exclusively in the category. Classification is also based on the
complement of cas genes that are present. Most CRISPR-Cas systems have a Cas1
protein. The phylogeny of Cas1 proteins generally agrees with the classification
system, but exceptions exist due to module shuffling.Many organisms contain
multiple CRISPR-Cas systems suggesting that they are compatible and may share
components. The sporadic distribution of the CRISPR-Cas subtypes suggests that
the CRISPR-Cas system is subject to horizontal gene transfer during
microbial evolution.
Class Cas Cas Signature Function
type subtype protein
1 I — Cas3 Single-stranded DNA nuclease (HD domain) and
ATP-dependent helicase

I-A Cas8a, Cas5 Cas8 is a Subunit of the interference module that is

important in targeting of invading DNA by
recognizing the PAM sequence. Cas5 is required for
processing and stability of crRNAs

I-B Cas8b

I-C Cas8c

I-D Cas10d contains a domain homologous to the palm domain

of nucleic acid polymerases and nucleotide cyclases

I-E Cse1, Cse2

I-F Csy1, Csy2, Type IF-3 have been implicated in CRISPR-

Csy3 associated transposons

I-G[Note 1] GSU0054

III — Cas10 Homolog of Cas10d and Cse1. Binds CRISPR target

RNA and promotes stability of the interference
complex

III-A Csm2 Not Determined

III-B Cmr5 Not Determined

III-C Cas10 or Csx11

III-D Csx10

III-E

III-F

IV — Csf1

IV-A

IV-B

IV-C

2 II — Cas9 Nucleases RuvC and HNH together produce DSBs,

and separately can produce single-strand breaks.
Ensures the acquisition of functional spacers during
adaptation.

II-A Csn2 Ring-shaped DNA-binding protein. Involved in

primed adaptation in Type II CRISPR system.

II-B Cas4 Endonuclease that works with cas1 and cas2 to

 generate spacer sequences

II-C Characterized by the absence of either Csn2 or Cas4

V — Cas12 Nuclease RuvC. Lacks HNH.

V-A Cas12a (Cpf1) Auto-processing pre-crRNA activity for multiplex

gene regulation

V-B Cas12b (C2c1)

V-C Cas12c (C2c3)

V-D Cas12d (CasY)

V-E Cas12e (CasX)

V-F Cas12f (Cas14,

C2c10)

V-G Cas12g

V-H Cas12h

V-I Cas12i

V-K[ Cas12k (C2c5) Type V-K have been implicated in CRISPR-associated

transposons

V-U C2c4, C2c8,

C2c9

VI — Cas13 RNA-guided RNase

VI-A Cas13a (C2c2)

VI-B Cas13b

VI-C Cas13c

VI-D Cas13d

VI-X Cas13x.1 RNA dependent RNA polymerase, Prophylactic RNA-

virus inhibition

VI-Y
Evolution
The cas genes in the adaptor and effector modules of the CRISPR-Cas system are
believed to have evolved from two different ancestral modules. A transposon-like
element called casposon encoding the Cas1-like integrase and potentially other
components of the adaptation module was inserted next to the ancestral effector
module, which likely functioned as an independent innate immune system. The
highly conserved cas1 and cas2 genes of the adaptor module evolved from the
ancestral module while a variety of class 1 effector cas genes evolved from the
ancestral effector module. The evolution of these various class 1 effector module
cas genes was guided by various mechanisms, such as duplication events. On the
other hand, each type of class 2 effector module arose from subsequent
independent insertions of mobile genetic elements. These mobile genetic
elements took the place of the multiple gene effector modules to create single
gene effector modules that produce large proteins which perform all the
necessary tasks of the effector module. The spacer regions of CRISPR-Cas systems
are taken directly from foreign mobile genetic elements and thus their long term
evolution is hard to trace. The non-random evolution of these spacer regions has
been found to be highly dependent on the environment and the particular foreign
mobile genetic elements it contains.
CRISPR-Cas can immunize bacteria against certain phages and thus halt
transmission. For this reason, Koonin described CRISPR-Cas as
a Lamarckian inheritance mechanism.[ However, this was disputed by a critic who
noted, "We should remember [Lamarck] for the good he contributed to science,
not for things that resemble his theory only superficially. Indeed, thinking of
CRISPR and other phenomena as Lamarckian only obscures the simple and
elegant way evolution really works". But as more recent studies have been
conducted, it has become apparent that the acquired spacer regions of CRISPR-
Cas systems are indeed a form of Lamarckian evolution because they are genetic
mutations that are acquired and then passed on. On the other hand, the
evolution of the Cas gene machinery that facilitates the system evolves through
classic Darwinian evolution.
Coevolution[edit]
Analysis of CRISPR sequences revealed coevolution of host and viral genomes.
Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR-
Cas-mediated gene regulation may contribute to the regulation of endogenous
bacterial genes, particularly during interaction with eukaryotic hosts. For
example, Francisella novicida uses a unique, small, CRISPR-Cas-associated RNA
(scaRNA) to repress an endogenous transcript encoding a
bacterial lipoprotein that is critical for F. novicida to dampen host response and
promote virulence.
The basic model of CRISPR evolution is newly incorporated spacers driving phages
to mutate their genomes to avoid the bacterial immune response, creating
diversity in both the phage and host populations. To resist a phage infection, the
sequence of the CRISPR spacer must correspond perfectly to the sequence of the
target phage gene. Phages can continue to infect their hosts' given point
mutations in the spacer.Similar stringency is required in PAM or the bacterial
strain remains phage sensitive.

Applications
CRISPR gene editing
CRISPR technology has been applied in the food and farming industries to
engineer probiotic cultures and to immunize industrial cultures (for yogurt, for
instance) against infections. It is also being used in crops to enhance yield,
drought tolerance and nutritional value.

 By the end of 2014, some 1000 research papers had been published that
mentioned CRISPR. The technology had been used to functionally inactivate
genes in human cell lines and cells, to study Candida albicans, to
modify yeasts used to make biofuels, and genetically modify
crop strains. Hsu and his colleagues state that the ability to manipulate the
genetic sequences allows for reverse engineering that can positively affect
biofuel production. CRISPR can also be used to change mosquitoes so they
cannot transmit diseases such as malaria.] CRISPR-based approaches
utilizing Cas12a have recently been utilized in the successful modification of
a broad number of plant species.
 In July 2019, CRISPR was used to experimentally treat a patient with a
genetic disorder. The patient was a 34-year-old woman with sickle cell
disease.
 In February 2020, progress was made on HIV treatments with 60–80% of
the integrated viral DNA removed in mice and some being completely free
from the virus after edits involving both LASER ART, a new anti-retroviral
therapy, and CRISPR.
 In March 2020, CRISPR-modified virus was injected into a patient's eye in an
attempt to treat Leber congenital amaurosis.
In the future, CRISPR gene editing could potentially be used to create new species
or revive extinct species from closely related ones.
CRISPR-based re-evaluations of claims for gene-disease relationships have led to
the discovery of potentially important anomalies.
 In July 2021, CRISPR gene editing of hiPSC's was used to study the role of
MBNL proteins associated with DM1.
The CRISPR technique has a positive response in working towards different
disorders like Nervous system, Circulatory system, Stem cells, blood disorders,
muscular degeneration. This tool has made advanced approaches in both
therapeutic and biomedical systems and some of the applications are discussed
below,

 1.1 β-Hemoglobinopathies
This disease comes under genetic disorders which are caused by mutation
occurring in the structure of hemoglobin or due to substitution of different
amino acids in globin chains. Due to this, the red blood cells (RBC) cause a
string of obstacles such as failure of heart, hindrance of blood vessels,
defects in growth and optical problems. To rehabilitate β-
hemoglobinopathies, the patient's multipotent cells are transferred in a
mice model to study the rate of gene therapy in ex-vivo which results in
expression of mRNA and the gene being rectified. Intriguingly RBC half-life
was also increased.
 1.2 Hemophilia
It is a loss of function in blood where clotting factors do not work properly.
There are two types, Hemophilia A and Hemophilia B. By using CRISPR-
Cas9, a vector is inserted into bacteria. The vector used is Adenoviral vector
which helps in correction of genes. Doubtlessly, CRISPR has given hope for
the treatment of hemophilia by setting right the genes.

 1.3 Neurological disorders

CRISPR is used in suppressing the mutations which cause gain of function

and also repairs the mutations with loss of functions with gene editing in
neurological disorders.[196] The gene editing tool setup a foothold in vivo
application for assimilation of molecular pathways.
  1.4 Blindness
The Eye disorders became more impediment for the doctors to treat the
victims. Moreover, the retinal tissue present in the eye is free from body
immune response. The most commonly occurring worldwide eye diseases
are cataract and retinitis pigmentosa (RP). These are caused by a missense
mutation in the alpha chain that leads to permanent blindness. The
approach of CRISPR is to bag the gene coding retinal protein and edit the
genome which results in good vision.

 1.5 Cardiovascular diseases

The CRISPR technology works more efficiently towards diseases related to
the heart. Due to deposition of cholesterol in the
walls of the artery leads to blockage of flow of blood. This is caused by
mutation in low density lipoprotein cholesterol receptors (LDLC) which
results in release of cholesterol into blood in higher levels. This can be
treated by deletion of base pair in exon 4 of LDLC receptor. This is a
nonsense mutation.