CRISPR Cas9 endonuclease of the Type II CRISPR/Cas system


A genome modifying tool, the prokaryotic type II clustered
regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is swiftly
modernizing the genetic engineering field, altering the genomes of a large
variety of organisms. Cancer genetics can be changed using experimental
approaches based on this multipurpose technology 1. Principal component Cas9 endonuclease of the Type II
CRISPR/Cas system is a prokaryotic adaptive restriction system against invading
nucleic acids, such as those of bacteriophages and plasmids. Recently, this
RNA-directed DNA endonuclease has been coupled to target DNA sequences of
interest. This system finds and cuts target protospacer DNA
precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif) through
guidance of a 20 nucleotide RNA (gRNA),   2. Cas9 not only edits the genomes of a number of
different prokaryotic and eukaryotic species, but also an effective system for
site-specific transcriptional repression or activation 3. It is simple, precise, and worthwhile
means of genome wide gene editing through modification of any genomic sequences,
equivalent to the search function found in modern word processors, Through
short RNA search string the Cas9 can be guided to specific locations within
complex genomes. Cas9-mediated genetic perturbation is simple and accessible, exposing
the functional organization of the genome at the systems level and establishing
causal linkages between genetic variations and biological phenotypes 4 As a RNA-guided
DNA recognition platform, it permits precise, scalable and vigorous RNA-guided
transcription regulation 5, as compared to RNA-mediated
interference (RNAi), that uses small interfering RNAs (siRNAs) or short hairpin
RNAs (shRNAs), that act as sequence-specific gene suppression in eukaryotic
organisms 6 but also non-specific
and inefficient 7. Genome engineering via RNA-guided CRISPR-Cas9 system
provides a novel methodology, to induce genomic modifications under the
endogenous gene promoters 8. Targeted
genome editing choices has been modernized using CRISPR-Cas9 in past few years 9. These programmable RNA-guided
endonucleases (RGENs) comprise two RNA elements, CRISPR RNA (cRNA) and its
transactivating RNA (tracRNA), which fused together and induce a targeted
double-strand break (DSB),providing a corresponding DNA template, any specific
gene sequence can be introduced via homologous recombination (HR) 10.

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CRISPR/Cas is a microbial adaptive immune system that
uses a single-guide RNA to target the Cas9 nuclease to cleave a specific
genomic sequence. Double-stranded DNA breaks induced by Cas9 are either repaired
by imperfect non-homologous end joining or by homology-directed repair that produce
insertions or deletions (indels). The CRISPR/Cas9 system is a powerful tool for
genome engineering in various species due to its specificity, simplicity and flexibility
11. Cancer can be cured based on CRISPR screen reports on
various cancer cell lines

12. Acute myeloid leukemia (AML), a human malignancy disorder
with long term survival rate of less than 30% needs additional therapies (Ferrara and Schiffer, 2013). Steady progress
in decoding its molecular pathogenesis has been made over the last few decades
with a dramatic acceleration in recent years, particularly as a consequence of
advances in cancer genomics (Cancer Genome
Atlas Research
Network, 2013; Welch et al., 2012).


How to construct CRISPR

complementary genomic sequences Double stranded break (DSB) are induce and
hybridize by single guide RNA (sgRNA) Cas9 endonuclease of the type II CRISPR
system 13. The protospacer-adjacent
motif (PAM) is a short DNA sequence adjacent to the RNA-binding site , self
from non-self can be differentiated by CRISPR Cas mechanism (17); Through
sequence complementarity the post-transcriptional processing and maturation of
the CRISPR RNA (crRNA) is directed by the small 
transactivating CRISPR RNA (tracrRNA) (18); and the CRISPR–Cas9 system
from S. thermophilus can function in Escherichia coli and provide resistance
against foreign plasmids14. Analysis about CRISPR–Cas9 biology, suggest that the
Streptococcus pyogenes Cas9 protein can bind to a tracrRNA–crRNA complex or to
a designed, chimeric sgRNA to generate a double-strand break (DSB) at a
specific site of the target DNA in vitro 13, 15. Similarly another report revealed that to cut DNA S.
thermophilus Cas9 could interact with the tracrRNA–crRNA complex 13. This seminal observation allowed the rapid  use of Cas9 and RNAs for in vivo genome
editing 16.


The short
repetitive stretches of DNA in bacterial genome are separated by spacers. These
spacers are often composed of bits of foreign DNA that has role in bacterial
molecular memory of prior infection. When the same pathogen is encountered
again, the stretches of repeats and spacers are transcribed to form CRISPR RNAs
(crRNA). Along with transactivating RNA (tracrRNA), it forms a kind of GPS
system for a series of CRISPR-associated (Cas) proteins that function like
molecular scissors, destroying the invader’s genome targeted DNA sequence.



Transcription repression by CRISPRi

RNAi Synthesis machinery is absent in bacteria,
and targeted gene regulation platforms are limited in bacteria. In sequence-specific
gene repression use of  dCas9 was first
demonstrated in E. coli as a technology called CRISPR interference (CRISPRi) 5. RNA polymerase (Pol) can be blocked by
interference of transcription elongation by the pairing of dCas9 with a
sequence-specific sgRNA, forming dCas9–sgRNA complex. It can also block transcription initiation by
disrupting transcription factor binding 17, 18. Efficient
dCas9-mediated transcription repression in bacteria allows the possibility of
using RNA-guided mechanisms for transcription repression and activation in
diverse organisms 19. CRISPR–Cas is currently divided into two major classes and
five types, of which type II is the most widely used for genome-engineering
applications 17.



Transcription activation by CRISPRa


CRISPR-mediated gene activation, called
CRISPRa, uses dCas9 fusion proteins to recruit transcription activators. For E.coli gene activation the holoenzyme
is assembled at targeted promotor site through the fusion of
dCas9 with the ?-subunit of the E. coli Pol 20. Currently only few reports are
availaible on CRISPRa in bacteria, but more work is needed to achieve robust
and consistent gene activation in bacteria 5. Several genes can be targeted
simultaneously through CRISPR–dCas9 by using multiple sgRNAs. Recently, by using
scaffold RNAs (scRNAs) a method for simultaneous activation and repression of
genes was established 21.


22 In acute myeloid leukemia (AML) additional
therapeutic targets are identified by researchers, they identify genetic
vulnerabilities in AML cells  by
optimizing a genome-wide clustered regularly interspaced short palindromic repeats
(CRISPR) screening platform. They identified 492 AML-specific cell-essential genes,
containing several established therapeutic targets such as BCL2, MEN1, and DOT1L,
and many other genes comprising clinically actionable candidates, using pharmacological
and genetic inhibition validated selected genes, and for downstream study selected
KAT2A as a candidate. KAT2A inhibition promotes anti-AML activity by inducing
myeloid differentiation and apoptosis, and suppressed the growth of primary
human AMLs of diverse genotypes while sparing normal hemopoietic
stem-progenitor cells, results proposed that therapeutic strategy in AML can be
investigated through KAT2A inhibition that provide a great genetic
vulnerabilities of AML to pursued in downstream studies.

23  Using the IDH2 R140Q mutation as a model, AML
associated mutations in or from human leukemic cells can be removed or
reproduced by using the RNA guided clustered regularly interspaced short
palindromic repeats (CRISPR)-Cas9 system via homologous recombination or via introduction of a DNA template at
the endogenous gene locus. Through precise modelling, AML development and
progression can be estimated that provide basis for future therapeutic


Digestive enzymes Isocitrate
dehydrogenases (IDHs) catalyse the oxidative decarboxylation of isocitrate, generating
alpha-ketoglutarate (a-ketoglutarate) and CO2. In AML patients there are
frequent mutations in IDH1/2 genes, and IDH2 R140Q is the most frequent IDH
mutation in AML 24. In a transgenic
mouse model IDH2 R140Q is a key driver mutation, supporting its significance for
the treatment of human AML therapeutic target 25.

Despite of enormous
research into acute myeloid leukemia (AML) during last few years, the majority
of AML patients still die from their disease. So, there should be new AML
therapies. AML is a heterogeneous disease sheltering a multitude of genetic and

and it is highly likely that the various AML subtypes require different
targeted therapeutic approaches 23.