Abstract gene and IDH1/2 genes. Mutation in these

Abstract

Acute myeloid leukemia (AML) is blood cell cancer, categorized by the rapid proliferation
of abnormal or defected cells that are formed in the bone marrow and blood.
Though AML was not curable about 50 years ago but now researchers have
identified many new therapeutic drug targets by using prokaryotic type II clustered regularly interspaced
short palindromic repeats (CRISPR)
that are DOT1L, BCL2, and MEN1. Other new therapeutic targets are KAT2A
gene and IDH1/2 genes. Mutation in these genes causes AML in patients. These
mutations can be removed by using CRISPR Cas9 and mutation in these genes can
be induced through CRISPR to
create disease models to study and cure AML effectively. The emerging
technology CRISPR can be used as screening tool for use in functional genomics
to find out new targets to cure Acute
myeloid leukemia (AML).

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Introduction

A genome modifying tool, 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. Prokaryotic Type II CRISPR/Cas system’s principal component
is Cas9 endonuclease that restrict the invading organisms nucleic acid i.e.
bacteriophages and plasmids. Nowadays, this RNA guided DNA endonuclease system
target specific DNA sequence. 20 nucleotide RNA (gRNA) guide to
cut 3 base pairs upstream target protospacer DNA accurately of a PAM
(Protospacer Adjacent Motif) 2. Cas9 edits the genomes different prokaryotic and
eukaryotic species, but also an effective system for site-specific
transcriptional repression or activation 3.

20
nucleotide RNA (gRNA) guide to cut 3 base pairs upstream target protospacer DNA
accurately of a PAM (Protospacer Adjacent Motif)

 

Prokaryotic Type II CRISPR/Cas system’s principal component
is Cas9 endonuclease that restrict the invading organisms nucleic acid i.e.
bacteriophages and plasmids.

 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.

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 13. 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 14.

Construction of CRISPR cassette

At complementary genomic sequences Double stranded
break (DSB) are induce and hybridize by single guide RNA (sgRNA) Cas9
endonuclease of the type II CRISPR system
15. 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 plasmids16. 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 15, 17. Similarly another report revealed that to cut DNA S.
thermophilus Cas9 could interact with the tracrRNA–crRNA complex 15. This seminal observation allowed the rapid  use of Cas9 and RNAs for in vivo genome editing
18.

 

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.

Repression of Transcription by CRISPRi

The machinery of
RNAi is not present in bacteria and thus the process of targeted gene
regulation is limited in bacteria. Initially, the significance of dCas9 for sequence-specific gene
repression was demonstrated in E. coli as a technology called CRISPR
interference (CRISPRi) 5. By pairing dCas9 with a sequence-specific
sgRNA, the dCas9–sgRNA complex can interfere with transcription elongation by
blocking RNA polymerase (Pol). It can also impede transcription initiation by
disrupting transcription factor binding 19, 20. Efficient
dCas9-mediated transcription repression in bacteria demonstrated the
possibility of using RNA-guided mechanisms for transcription repression and
activation in diverse organisms 21. CRISPR–Cas is currently divided into 5 types and 2 major
classes, of which type II is the most widely used for genome-engineering
applications 19.

Activation of Transcription by CRISPRa

Activation
of genes carried CRISPRa uses dCas9 proteins to activate transcription
activators. In E.Coli genes are activated by fusion of dCas9 with the ?-subunit
of E. coli Polymerase, this fusion allow the holoenzyme  to bind with a target promoter to activate
transcription 22. Activity
of CRISPRa  in bacteria is not well
elucidated uptill now, more work is required 
to attain strong and consistent activation of genes in bacteria 5.

CRISPR–dCas9 system is capable of
targeting many genes at a time by using multiple small guided RNAs, now
scaffold RNAs (scRNAs) are being used to develop a method to study repression
and activation of genes simultaneously 23.

Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is defined as one of the aggressive
forms of cancer by bone marrow, blood and other tissues infiltration developed
by the rapid increase in hematopoietic system cells which are abnormally
differentiated, clonal, poorly differentiated (Döhner, Hartmut, Daniel J. Weisdorf, and Clara D. Bloomfield.
“Acute myeloid leukemia.” New England Journal of Medicine373,
no. 12 (2015): 1136-1152.).

Therapeutic targets identified for Acute Myeloid Leukemia (AML) and
CRISPR

Many new
therapeutic drug targets were identified in acute myeloid leukemia (AML)
by  24 they developed (CRISPR) based screening method
for identification of genetic defects in acute myeloid (AML) cells. They found
492 cell-essential genes and AML specified genes, including many other approved
therapeutic targets such as DOT1L, BCL2, and MEN1, and many new genes were
found as clinically approachable targets, some selected genes were
authenticated by using genetic and pharmacological inhibition.

KAT2A gene

 KAT2A gene inhibition exhibit anti-AML
activity by causing induction of myeloid cell’s differentiation and apoptosis,
and suppression of growth of primary human Acute myeloid leukemia cells with
diversified genotypes while sparing normal hemopoietic stem-progenitor cells.
Results of this study suggested that inhibition of KAT2A gene must be explored
as a therapeutic approach in AML treatment.

IDH2 R140Q mutation

25  IDH2 R140Q mutation can be used as a model
which is an effective practice of using the (CRISPR)-Cas9 system guided by RNAs
to produce or remove mutations related to AML in or from human leukemic cells,
correspondingly, By introducing of a DNA
template at the endogenous gene locus via homologous recombination. Our
technology represents a precise way for AML
modeling to gain insights into AML
development and progression and provides a basis for future therapeutic
approaches. Isocitrate dehydrogenases (IDHs) are also known as digestive
enzymes they catalyse the production of alpha-ketoglutarate (a-ketoglutarate)
and CO2 from oxidative decarboxylation of isocitrate, AML
patients have a frequent history of mutations in IDH1/2 genes, but mutation
named as IDH2 R140Q is the most common IDH mutation in AML
26. IDH2 R140Q is
considerd to be a key driver mutation in the 
transgenic mouse models, and hence can be used as therapeutic target for
the treatment of AML in humans 27. Many patients
still die due to AML, In spite of
all the intense research into acute myeloid leukemia (AML)
during a past few decades. Thus, it is the need of hour to have many new AML therapies. This heterogeneous disease (AML) harbors an assembly of genetic and epigenetic
variations, and Many of the AML subtypes need diversified targeted therapeutic
approaches 25.

Steps involved in curing AML by
CRISPR

1.     
Introducing
IDH2 R140Q Mutation in Human Myeloid Leukemia Cells,

2.     
Propagation
and Epigenetic Changes due to IDH2 R140Q mutation in Human Myeloid Leukemia
Cells

3.     
Removal
of IDH2 R140Q Mutation from the Primary AML Patient Blasts by CRISPR Cas 9
system.

1.    
Introducing
IDH2 R140Q Mutation in Human Myeloid Leukemia Cells

 

Human myeloid leukemia cell line K562 lacks the IDH2 R140Q
mutation was selected for study. Three small-guide (sg) RNAs were selected to
target IDH2.  DNA templates selected for
targeted incorporation by HR after intended Double stranded breaks at IDH2 gene
locus was engineered and cloned in pBS-SK(+) vector to perform PCR-based
site-directed mutagenesis. The binding site for sgRNA at the IDH2 sequence was
introduced with Silent point mutations. DNA template carrying a red
fluorescence protein (RFP) fuse with the mutated IDH2 sequence by a 2A peptide.
Thus the cells with correct and successful insertion of DNA template at the
gene locus IDH2, the gene promoter causes the regulation of the incorporated
IDH2 R140Q mutation along with RFP expression. Thus, targeted cells were carefully
chosen by fluorescence-activated cell sorting (FACS) method.

 

2. Propagation and Epigenetic Changes
due to IDH2 R140Q Mutations in Human Myeloid Leukemia Cells

 

Unusually,
the cells K562 with IDH2 R140Q mutation were come up with enhanced in vitro
colony forming capacity as compared to the controls having wild type IDH2
induced analogously. Furthermore, K562 cells with induced IDH2 R140Q point
mutation exhibit a significant increase in H3K9me2, H3K27me2, and H3K4me3 after
successful genome editing by CRISPR Cas 9.

 

3. CRISPR-Cas9-Mediated
Removal of IDH2 R140Q Mutation from Primary AML Patient Blasts

 

Myeloid blasts showed an IDH2 R140Q mutation in a 66-year-old patient
with AML subtype FAB M4. To Eradicate mutations from the patient’s AML cells by
substituting the mutated IDH2  sequence
with normal IDH2 wt sequence, lentivirally transduced the primary blasts along
with Lenti CRISPRv2 carrying an sgRNA targeted for IDH2 R14Q. sgRNA sequences
were modified accordingly as the  point
mutation of the IDH2. Primary AML blasts taken from same AML patient
transfected by the similar method but introducing the IDH2 R140Q mutation
again. Because lentiviral is the major issue for specified genome editing by
using lentiviral transduction approaches.

The insertion of the normal wt IDH2 sequence in mutated cells and
in the control cells, correspondingly, was examined and conformed by using
targeted integration PCR along with by sequencing of the genomic DNA 25.

CRISPR-Cas9 as a
screening tool

The CRISPR-Cas9 system can be modified as a screening tool for use
in functional genetics study as pooled guide RNA
(gRNA) libraries, this technology may screens for new genes which upon
inactivation may show resistance to the toxins, chemotherapeutic drugs, and
directed treatments of cancer can be effectively led
28.For increasing CRISPR-Cas9
competence, gRNA scaffold developed for 
CRISPR imaging was tested 29 and results showed in recent reports that guide RNAs with  upgraded or modified scaffolds show
greater  knockout efficiency than the
guide RNAs with conventional scaffolds 30. Furthermore, an optimal
gRNA library was established by redesigning gRNAs for the genome of mouse by
using a newly designed pipeline (version 2 v2) that consist of 90,230 gRNAs
specifically targeting a total of 18,424 genes.

 

Genome-wide
dropout screening was performed in five AML
cell lines (MOLM-13, MV4-11, HL-60, OCIAML2, and OCI-AML3)
and in the fibrosarcoma cell line HT-1080 as a second non-AML reference. There is a small fraction of cells exhibiting no Cas9 activity in the bulk
of Cas9-expressing cells, but single-cell
cloning technique eradicate this fraction 
and exhibited uniform Cas9 activity. Selected clones transduced along
with human CRISPR library were collected to find out their gRNA content after
30 days culturing. The dropout competence of many identified cell-essential
genes was compared in accordance with the number of copies of chromosomes upon
which these genes are present and it is found that there is no major difference
that shows disruption of genes effectively regardless of copy number in AML
cell lines by Cas9. The gRNAs specificallly targeting the exons sequence
upstream of the MLL breakpoint region may disturb the cancer causing genes such
as MLL-AF9 and MLL-AF4 and they may get depleted in both MOLM-13 and MV4-11
cell lines. MLL is also acknowledged as KMT2A.
Furthermore, guide RNAs against FLT3 and NRAS exhibited specified deletion of
these mutated genes in the cell lines, however NPM1 was deleted in four of the
five AML cell lines including OCI-AML3 31.

Conclusion

A genome editing tool, the prokaryotic type II clustered
regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a new
and emerging technology for curing Acute myeloid leukemia (AML). In this review CRISPR
technology is used to cure AML by inhibition of genes involved in causing this
disease. The genes involved in AML are DOT1L, BCL2, and MEN1. Many new
therapeutic targets such as KAT2A gene and IDH1/2 gene were manipulated by
using CRISPR Cas9. Inhibition of both genes showed effective results in curing
disease, this suggest that these gene can be effective therapeutic drug targets
in future. CRISPR Cas9 is not only used for repression or inhibition of these
genes but it can also be used to create mutations in targeted genes to create
disease models. Now a day these kinds of disease models are being produce to
study cure of AML by CRISPR Cas9.

Future prospect

The CRISPR-Cas9 system can be used as a screening tool for use in
functional genetics study to find out many new genes involved in AML disease or
in other cancers.  Guide RNAs can be modified
to increase capability of CRISPR Cas9 for better gene knockdown. This rapidly emerging
technology can be used for repression and activation of transcription process
of genes so it can play a more effective in clinical research and in cancer
biology.